Nitrogen fixation using refactored nif clusters

ABSTRACT

The invention relates to methods for promoting fixed nitrogen from atmospheric nitrogen, and related products. Endophytic bacteria having an exogenous nif cluster promote fixed nitrogen for cereal plants.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/766,122, entitled “NITROGEN FIXATION USING REFACTORED NIF CLUSTERS,”filed Apr. 5, 2018, which is a national stage filing under U.S.C. § 371of PCT International Application PCT/US2016/055429, entitled, “NITROGENFIXATION USING REFACTORED NIF CLUSTERS,” filed Oct. 5, 2016, whichclaims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication No. 62/237,426, entitled “NITROGEN FIXATION IN SALMONELLAUSING REFACTORED NIF CLUSTERS”, filed Oct. 5, 2015, which are hereinincorporated by reference herein in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under IOS1331098 awardedby the National Science Foundation. The government has certain rights inthe invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(M065670369US02-SEQ-HCL.xml; Size: 119,626 bytes; and Date of Creation:Aug. 25, 2022) is herein incorporated by reference in its entirety.

BACKGROUND OF INVENTION

Availability of nitrogen is one of the principal elements limitinggrowth and development of crops, particularly in agricultural soils forplant production of food, feed, fiber and fuel. Excessive use ofsynthetic fertilizer to meet the food demands of growing populationposes an environmental threat in that it can cause harmful algal bloomsand disrupt beneficial soil microbial community [1]. On the other hand,over-farming in many developing countries with a scant supply offertilizer damages agricultural land and makes small farmers suffer fromthe poor yield of their crops [2].

Successful endophytic colonization of plants by human-pathogenicbacteria such as Salmonella enterica, Pseudomonas aeruginosa,Burkholderia cepacia, Escherichia coli O157:H7 has been demonstrated[3-5]. Salmonella can recognize plants as a suitable host and colonizein root tissues of alfalfa and barley [6,7].

SUMMARY OF INVENTION

The invention, in various aspects, relates to a method for providingfixed nitrogen from atmospheric nitrogen, comprising delivering amodified bacteria having an exogenous nif cluster to a cereal plant, orto soil where a cereal plant or seed is growing or is to be planted,wherein the modified bacteria provides fixed nitrogen.

In some embodiments, the nif cluster is a native nif cluster. In someembodiments, the nif cluster is a refactored nif cluster.

In other embodiments, the modified bacteria is a gamma-proteobacteria.In some embodiments, the modified bacteria is a Salmonella typhimurium.

In some embodiments, the Salmonella typhimurium strain is selected fromSL1344, LT2, and DW01.

In other embodiments, the modified bacteria is a E. coli, optionally ofstrain H7:0157.

In other embodiments, the nif cluster is a Klebsiella wild-type nifcluster, a Pseudomonas Stutzi nif cluster, or a Paenibacillus cluster.In some embodiments, the nif cluster is a refactored nif clusters.

In some embodiments, the cereal plant is selected from wheat, rye,barley, triticale, oats, millet, sorghum, teff, fonio, buckwheat,quinoa, corn and rice.

In some embodiments, the invention further comprises an exogenous geneencoding a plant growth-stimulating peptide.

In some embodiments, the exogenous gene encoding the plantgrowth-stimulating peptide is regulated by a type 3 secretion system(T3SS).

In some embodiments, the plant growth stimulating peptide is directlydelivered into root or stem tissues.

Aspects of the invention include a method, comprising delivering amodified non-pathogenic bacteria having exogenous genes for enablingplant endosymbiosis to a cereal plant, or to soil where a cereal plantor seed is growing or is to be planted.

In some embodiments, the non-pathogenic bacteria is E. coli.

In some embodiments, the genes encode effectors or apparatus for asecretion system.

In other embodiments, the apparatus for a secretion system is type 3secretion system (T3SS).

Aspects of the invention include compositions comprising anagriculturally suitable or compatible carrier, and agamma-proteobacteria having an exogenous nif cluster present on or inthe agriculturally suitable or compatible carrier.

In some embodiments, the proteobacteria is a Salmonella typhimurium orE. coli.

In other embodiments, the nif cluster is a native nif cluster.

In some embodiments, the nif cluster is a refactored nif cluster.

In some embodiments, the invention further comprises an exogenous geneencoding a plant growth-stimulating peptide.

In some embodiments, the agriculturally suitable or compatible carrieris selected from the group consisting of seeds, seed coats, granularcarriers, soil, solid carriers, liquid slurry carriers, and liquidsuspension carriers. In other embodiments the agriculturally suitablecarrier includes a wetting agents, a synthetic surfactant, awater-in-oil emulsion, a wettable powder, granules, gels, agar strips orpellets, thickeners, microencapsulated particles, or liquids such asaqueous flowables or aqueous suspensions.

In other embodiments the exogenous nif cluster or gene includes acontroller. The controller may be a nucleic acid encoding an IPTGinducible T7 RNA polymerase. Alternatively the controller may be apartitioning system encoded by the two par operons (parCBA and parDE).In some embodiments the partitioning system is a RK2 par system.

A seed or seedling of a cereal plant having a modified bacteriaassociated with an external surface of the seed or seedling is providedin other aspects of the invention. In some embodiments the modifiedbacteria has an exogenous nif cluster.

In other aspects the invention is a cereal plant having a modifiedbacteria in the plant, wherein the modified bacteria has an exogenousnif cluster.

The nif cluster may be a native nif cluster or a refactored nif cluster.In some embodiments the nif cluster is a Klebsiella wild-type nifcluster, a Pseudomonas Stutzi nif cluster, or a Paenibacillus cluster.In some embodiments the modified bacteria is a gamma-proteobacteria suchas a Salmonella typhimurium, optionally a Salmonella typhimurium strainselected from SL1344, LT2, and DW01 or an E. coli, optionally of strainH7:0157.

The cereal plant in some embodiments is selected from wheat, rye,barley, triticale, oats, millet, sorghum, teff, fonio, buckwheat,quinoa, corn and rice.

Optionally the seed or seedling further includes an exogenous geneencoding a plant growth-stimulating peptide. The exogenous gene encodingthe plant growth-stimulating peptide, in some embodiments, is regulatedby a type 3 secretion system (T3SS).

In some embodiments the exogenous gene is in root or stem tissues of thecereal plant.

In some embodiments the modified bacteria may be provided in form ofsolutions, dispersions, sclerotia, gel, layer, cream, coating, or dip.

In some embodiments the plant, parts of plants or the area surroundingthe plants is selected from leaf, seed, branches, soil, stems, roots. Insome embodiments the modified bacteria is associated with (i.e. admixed,in physical contact with or present near) the plant, parts of plants orthe area surrounding the plants or is incorporated therein. In someembodiments the seeds are inoculated or coated with the modifiedbacteria. In certain embodiments, the modified bacteria is disposed inan amount effective to be detectable within a target tissue of themature agricultural plant selected from a fruit, a seed, a leaf, or aroot, or portion thereof.

In other embodiments, the plant, the seed or seedling comprises at leastabout 100 CFU, for example, at least about 200 CFU, at least about 300CFU, at least about 500 CFU, at least about 1,000 CFU, at least about3,000 CFU, at least about 10,000 CFU, at least about 30,000 CFU, atleast about 100,000 CFU or more, of the modified bacteria on itsexterior surface.

In another embodiment, the modified bacteria is disposed on an exteriorsurface or within a tissue of the plant, the seed or seedling in anamount effective to be detectable in an amount of at least about 100CFU, for example, at least about 200 CFU, at least about 300 CFU, atleast about 500 CFU, at least about 1,000 CFU, at least about 3,000 CFU,at least about 10,000 CFU, at least about 30,000 CFU, at least about100,000 CFU.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 shows nitrogenase activity in Salmonella strains. Nitrogenaseactivity of native and refactored nif clusters in diverse Salmonellastrains were measured by acetylene reduction assay. Non-detectableethylene production was indicated by asterisks.

FIG. 2 shows endophytic colonization of Zea mays B73 by entericbacteria. Internal colonization of maize roots by either S. typhi ATCC14028 or E. coli MG1655 (a control) was investigated. While there is noCFU of E. coli MG1655 from the crushed maize roots which is surfacesterilized, S. typhi ATCC 14028 cells were recovered from inside theroot tissues. Error bars represent standard deviation (n=6 for MG1655and n=10 for ATCC14028).

FIG. 3 shows ethylene production in maize plant seedlings. The box plotshows the distribution of ethylene production between the plantseedlings inoculated with the engineered S. typhi ATCC 14028 and thecontrol wild-type S. typhi ATCC14028 (no nif cluster). Dots representethylene production from individual plants in a group (n=33 (control),39 (native nif), 39 (refactored nif)). The box extends from 25% to 75%quartile. The central line represents the median of the ethyleneproduction in a group. The whiskers represent 75% quartile plus 1.5times the interquartile range (upper whiskers) and 25% quartile minus1.5 times the interquartile range (lower whiskers). Asterisk indicatesstatistically significant difference between the refactored nif and thecontrol group (Student's t-test, ***P<0.0001).

FIGS. 4A and 4B are graphs showing the stability of genetic systems inthe Salmonella strains obtained from the surface-sterilized roots. FIG.4A shows a controller device composed of a sensor, T7 RNA polymerase anda selective marker showed no loss from the genome-based expressionsystem. FIG. 4B shows the RK2 par system on the nif plasmid based on thepBBR1 origin of replication leads to an increase in the plasmidstability.

FIG. 5 shows a schematic of a controller for mini-Tn7 insertion(pR6K-T7RM).

DETAILED DESCRIPTION

Endophytic bacteria that are symbiotic with host plants can begenetically engineered to deliver proteins to the host and therebyregulate properties of plants. In non-cereal plants bacteria can be usedto provide fixed nitrogen, reducing the need for nitrogen richfertilizer. In cereal plants, however, bacterial systems for providingfixed nitrogen have never been developed despite many attempts over theyears to develop such systems. A method for manipulating endophyticbacteria such that they are capable of providing fixed nitrogen tocereal plants has been discovered according to the invention. Endophytesmay occupy the intracellular or extracellular spaces of plant tissue,including the leaves, stems, flowers, fruits, seeds, or roots.

The methods of the invention are useful for several purposes such asreducing fertilization needs, reducing fertilization pollution,providing an eco-friendly crop production, enhanced crop production,improved oil content in plants, improved protein content in plants, thereduction of nitrogen contamination of water, and the enrichment of thecarbon content relative to nitrogen and carbon in relation to a soil'sorganic phase.

A limiting factor for crop productivity of agricultural crops is thenitrogen content in soil and water. The supply of this element hasdwindled over time as crop demands increased. Nitrogen is one of theprimary nutrients essential to all forms of life, including plants.However, nitrogen must first be converted to a form that plants canutilize. Biological Nitrogen Fixation (BNF) is the conversion ofatmospheric nitrogen (N₂) to ammonia (NH₃) using the enzyme nitrogenase.This reaction consumes a tremendous amount of energy as N₂ contains atriple bond. The bond energy in a nitrogen molecule is about 225kcal/mol. Few BNFs are performed in nature as a result of a symbioticrelationship between plants and several bacterial species that make up a“nitrogenase enzymatic complex.”

The bacterial species that produce the nitrogenase enzymatic complexinclude diazotrophs such as cyanobacteria, azotobacteraceae, rhizobia,and frankia. However, only a few plant species can live in a symbioticrelationship with diazotrophs. For example, the pea plant from thelegume family lives in symbiosis with bacteria from the rhizobia family.In particular, rhizobia bacteria penetrate the pea plant's rootscreating root nodules that contain bacteria that fix nitrogen (toammonia) while the plant donates carbon (sugar). Improving either thesymbiosis, or extending the host range would therefore be beneficial forplant survival, but achieving this goal includes many challengesincluding the complexity of the process and lack of basic knowledge.

Biological nitrogen fixation is carried out by a complex of threeproteins (nitrogenase), encoded by nifH, rifD and nifK, which areassembled and activated by an additional 17 genes [8]. Transferring anif cluster to a new host is challenging because of the fact that thepathway is very sensitive to small changes in gene expression and theregulatory control in many organisms is not well established [8,9]. Asshown in the Examples, a refactoring method was applied to a 16 gene nifcluster from Klebsiella oxytoca M5a1 to engineer a system for regulatingnif. The method modularized the gene cluster into a set ofwell-characterized genetic parts. Refactoring can be used as a platformfor large-scale part substitutions that facilitate the swapping ofregulation to that which will function in a new host. Refactoring alsois valuable in eliminating the response to signals that repress thenative nif cluster, including ammonia and oxygen.

Quite surprisingly, it was discovered that nif clusters, both wild typeand refactored nif, transferred into endophytic bacteria enable thebacteria to provide fixed nitrogen in cereal plants. This is the firstdemonstration that the transfer of native and synthetic nif clustersinto endophytic bacteria can be used to provide fixed nitrogen to crops.The experiments presented in the Examples below demonstrate that geneticsensors connected to refactored nif clusters successfully regulatednitrogen fixation pathway at three different Salmonella strains inresponse to a chemical signal. The refactored nif clusters allows thetesting of large populations of enteric bacteria isolated from plantsfor efficient symbiosis that delivers nitrogen to crops.

Synthetic nucleic acids encoding wild type and refactored nif clusterscan be used to produce genetically modified bacteria. The modifiedbacteria useful according to the invention are endophytes which areendosymbionts. Endosymbionts do not cause apparent disease in plants forsome or all of its life cycle. Bacterial endophytes may belong to abroad range of taxa, including α-Proteobacteria, β-Proteobacteria,γ-Proteobacteria, Firmicutes, and Actinobacteria. It is particularlypreferred according to methods of the invention to use γ-Proteobacteria.

In some embodiments, examples of endophytic bacteria that areγ-Proteobacteria include but are not limited to Salmonella spp.,Yersinia pestis, Vibrio cholerae, Pseudomonas aeruginosa, Escherichiacoli, Xanthomonas axonopodis pv. citri and Pseudomonas syringae pv.actinidiae. In preferred embodiments γ-Proteobacteria include Salmonellaand Escherichia coli.

The modified bacteria of the invention, are used to promote fixednitrogen from atmospheric nitrogen. The term “plant” as used hereinrefers to cereal plants. The term includes all parts of a plant such asgerminating seeds, emerging seedlings and vegetation including all belowground portions (such as the roots) and above ground portions. Cerealsare the cultivated forms of grasses (Poaceae) and include for examplewheat (inclusive spelt, einkorn, emmer, kamut, durum and triticale),rye, barley, rice, wild rice, maize (corn), millet, sorghum, teff, fonioand oats. The term cereal plants also includes pseudocereals, such asamaranth, quinoa and buckwheat.

Additionally, the modified bacteria can be genetically engineered todeliver other factors such as plant growth-stimulating peptides directlyinto root or stem tissues. For instance, genes expressing proteins thataffect plants can be engineered into a type 3 secretion system (T3SS).Synthetic control will be able to be regulated by expressing of T3SS inbacteria. Methods of engineering bacteria in this manner are describedin Widmaier, D. M. et al. [3].

Thus, the methods according to the invention can also involvegenetically modifying bacteria to further treat the cereal plants. Theterm “genetically modified bacteria” refers to bacteria whose geneticmaterial has been modified by the use of recombinant DNA techniques toinclude an inserted sequence of DNA that is not native to that bacterialgenome or to exhibit a deletion of DNA that was native to that species'genome. Often, a particular genetically modified bacteria will be onethat has obtained its genetic modification(s) by a recombinant DNAtechnique. Typically, one or more genes have been integrated into thegenetic material of a genetically modified bacteria. The gene may beinserted into the T3SS region.

A nif cluster is a collection of genes encoding enzymes involved in thefixation of atmospheric nitrogen into a form of nitrogen available toliving organisms. The primary enzyme encoded by the nif genes is thenitrogenase complex which is in charge of converting atmosphericnitrogen (N₂) to other nitrogen forms such as ammonia which the organismcan use for various purposes. Besides the nitrogenase enzyme, the nifgenes also encode a number of regulatory proteins involved in nitrogenfixation. The nif genes are found in both free-living nitrogen-fixingbacteria and in symbiotic bacteria associated with various plants. Theexpression of the native nif genes are induced as a response to lowconcentrations of fixed nitrogen and oxygen concentrations (the lowoxygen concentrations are actively maintained in the root environment ofhost plants). Refactored nif clusters can be designed to be regulated byexogenous factors and/or constitutively regulated.

As used herein, a “genetic cluster” refers to a set of two or more genesthat encode gene products. A target, naturally occurring, or wild typegenetic cluster is one which serves as the original model for therefactoring. In some embodiments, the gene products are enzymes. In someembodiments, the gene cluster that is refactored is the nif nitrogenfixation pathway.

Each genetic cluster is organized into transcriptional units which arecomposed of a plurality of modular units. A modular unit is a discreetnucleic acid sequence that is made up of one or more genetic components.A genetic component may include anything typically found in a geneticfragment. For instance a genetic component incudes but is not limited togenes, regulatory elements, spacers, non-coding nucleotides. Some or allof these are found within each modular unit. Within the modular unit oneor more of the synthetic regulatory elements may be genetically linkedto one or more protein coding sequences of the genetic cluster.

While multiple modular units may be composed of the same gene andregulatory elements, the units may differ from one another in terms ofthe orientation, position, number etc. of the gene and regulatoryelements. Other modular units may have some elements in common withother modular units but include some different elements. Yet othermodular units may be completely distinct and do not overlap with othermodular units. The great diversity of the modular units is what leads tothe diversity of the assembled genetic clusters in a library.

The modular units within the genetic cluster are arranged such that theplurality of distinct non-naturally occurring genetic clusters aredistinct from a naturally occurring genetic cluster based on the number,the order, and/or the orientation of particular genetic components. Thenumber of genetic components within a modular unit may be easily varied.For instance, one modular unit may have a single promoter or terminator,whereas another modular unit may have 5 promoters and 2 terminators. Thevariation that may be achieved by manipulation of this factor issignificant. Additionally the order of the components within a modularunit may be varied dramatically. Multiple sets of modular units may begenerated where a single order of two components may be switched. Thisfactor would also generate significant diversity. Switching theorientation of a component in the modular unit is also a viable way ofgenerating diversity. While it may be expected that switching theorientation of one or more genetic components might interfere withfunctionality it has been demonstrated herein that genetic nif clustershaving different orientations are actually functional.

The refactoring process involves several levels of restructuring geneticclusters. For example, the codons of essential genes in a geneticcluster, such as the nif cluster, are changed to create a DNA sequencedivergent from the wild-type (WT) gene. This may be achieved throughcodon optimization. Recoded genes may be computationally scanned toidentify internal regulators. These regulatory components may then beremoved. They are organized into operons and placed under the control ofsynthetic parts (promoters, ribosome binding sites, and terminators)that are functionally separated by spacer parts. Finally, a controllerconsisting of genetic sensors and circuits that regulate the conditionsand dynamics of gene expression may be added.

The genetic components in the refactored genetic cluster typically willinclude at least one synthetic regulatory element. A syntheticregulatory element is any nucleic acid sequence which plays a role inregulating gene expression and which differs from the naturallyoccurring regulatory element. It may differ for instance by a singlenucleotide from the naturally occurring element. Alternatively it mayinclude one or more non-natural nucleotides. Alternatively it may be atotally different element. In each case, it may be considered to be anexogenous regulatory element (i.e. not identical to the naturallyoccurring version). Thus, a “regulatory element” refers to a nucleicacid having nucleotide sequences that influence transcription ortranslation initiation or rate, or stability and/or mobility of atranscription or translation product. Regulatory regions include,without limitation, promoter sequences, ribosome binding sites,ribozymes, enhancer sequences, response elements, protein recognitionsites, inducible elements, protein binding sequences, 5′ and 3′untranslated regions (UTRs), transcriptional start sites, transcriptionterminator sequences, polyadenylation sequences, introns, andcombinations thereof.

In some embodiments, the regulatory sequence will increase theexpression of a gene. In other embodiments, the regulatory sequence willdecrease the expression of a gene. In some embodiments the regulatorysequence may be a protein-binding sequence, for example a transcriptionfactor binding site. In some embodiments, the regulatory sequence may bea polymerase-binding site. In some embodiments, the regulatory sequenceis a terminator. The terminator may require an additional factor toindicated the end of the sequence for transcription, for example arho-dependent terminator. In some embodiments, a regulatory sequence isa sequence that binds a ribosome, such as a ribosome-binding site (RBS).In some embodiments, the regulatory sequence indicates where translationwill begin. It will be evident to one of ordinary skill in the art thatregulatory sequences differ in their strength of regulation. Forexample, there exist strong promoter sequences, gene expression fromwhich is higher than gene expression from a weak promoter sequence.Similarly, there exist strong RBS sequences that recruit and bindribosomes with higher affinity than a RBS sequence that is characterizedas weak. In some embodiments, the regulatory sequence may be aninducible or conditional regulatory sequence. In some embodiments, theregulatory sequence will exist 5′ or upstream of a protein-codingsequence. In other some embodiments, the regulatory sequence will exist3′ or downstream of a protein-coding sequence. In still otherembodiments, the regulatory sequence may be present within aprotein-coding sequence. Any given protein-coding sequence may beregulated by one or more regulatory sequences. Non-limiting examples ofregulatory sequences include the bacteriophage T7 promoter, sigma 70promoter, sigma 54 promoter, lac promoter, rho-dependent terminator,stem-loop/rho-independent terminator.

“Exogenous” with respect to a nucleic acid indicates that the nucleicacid is part of a recombinant nucleic acid construct, or is not in itsnatural environment. For example, an exogenous nucleic acid can be asequence from one species introduced into another species, i.e., aheterologous nucleic acid. Typically, such an exogenous nucleic acid isintroduced into the other species via a recombinant nucleic acidconstruct. An exogenous nucleic acid also can be a sequence that isnative to an organism and that has been reintroduced into cells of thatorganism. An exogenous nucleic acid that includes a native sequence canoften be distinguished from the naturally occurring sequence by thepresence of non-natural sequences linked to the exogenous nucleic acid,e.g., non-native regulatory sequences flanking a native sequence in arecombinant nucleic acid construct. In addition, stably transformedexogenous nucleic acids typically are integrated at positions other thanthe position where the native sequence is found. The exogenous elementsmay be added to a construct, for example using genetic recombination.Genetic recombination is the breaking and rejoining of DNA strands toform new molecules of DNA encoding a novel set of genetic information.

“Expression” refers to the process of converting genetic information ofa polynucleotide into RNA through transcription, which is catalyzed byan enzyme, RNA polymerase, and into protein, through translation of mRNAon ribosomes.

Promoters may be constitutive or inducible. Examples of constitutivepromoters include, without limitation, the retroviral Rous sarcoma virus(RSV) LTR promoter (optionally with the RSV enhancer), thecytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see,e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, thedihydrofolate reductase promoter, the β-actin promoter, thephosphoglycerol kinase (PGK) promoter, and the EF1α promoter[Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system [WO 98/10088]; theecdysone insect promoter [No et al, Proc. Natl. Acad. Sci. USA,93:3346-3351 (1996)], the tetracycline-repressible system [Gossen et al,Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], thetetracycline-inducible system [Gossen et al, Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)], the RU486-inducible system [Wang et al, Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)] and therapamycin-inducible system [Magari et al, J. Clin. Invest.,100:2865-2872 (1997)]. Still other types of inducible promoters whichmay be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly.

The regulatory elements may be in some instances tissue-specific.Tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.)are well known in the art. Exemplary tissue-specific regulatorysequences include, but are not limited to the following tissue specificpromoters: a liver-specific thyroxin binding globulin (TBG) promoter, aninsulin promoter, a glucagon promoter, a somatostatin promoter, apancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, acreatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, aα-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT)promoter. Other exemplary promoters include Beta-actin promoter,hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9(1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. GeneTher., 7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol.Biol. Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al.,J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J.Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cellreceptor α-chain promoter, neuronal such as neuron-specific enolase(NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15(1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc.Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgfgene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among otherswhich will be apparent to the skilled artisan.

In some instances the modular units or genetic clusters may be designedto lack in restriction recognition sites. Restriction endonucleasescleave DNA with extremely high sequence specificity and due to thisproperty they have become indispensable tools in molecular biology andmolecular medicine. Over three thousand restriction endonucleases havebeen discovered and characterized from a wide variety of bacteria andarchae. Comprehensive lists of their recognition sequences and cleavagesites can be found at REBASE.

As used herein the term “isolated nucleic acid molecule” refers to anucleic acid that is not in its natural environment, for example anucleic acid that has been (i) extracted and/or purified from a cell,for example, an algae, yeast, plant or mammalian cell by methods knownin the art, for example, by alkaline lysis of the host cell andsubsequent purification of the nucleic acid, for example, by a silicaadsorption procedure; (ii) amplified in vitro, for example, bypolymerase chain reaction (PCR); (iii) recombinantly produced bycloning, for example, a nucleic acid cloned into an expression vector;(iv) fragmented and size separated, for example, by enzymatic digest invitro or by shearing and subsequent gel separation; or (v) synthesizedby, for example, chemical synthesis. In some embodiments, the term“isolated nucleic acid molecule” refers to (vi) an nucleic acid that ischemically markedly different from any naturally occurring nucleic acid.In some embodiments, an isolated nucleic acid can readily be manipulatedby recombinant DNA techniques well known in the art. Accordingly, anucleic acid cloned into a vector, or a nucleic acid delivered to a hostcell and integrated into the host genome is considered isolated but anucleic acid in its native state in its natural host, for example, inthe genome of the host, is not. An isolated nucleic acid may besubstantially purified, but need not be. For example, a nucleic acidthat is isolated within a cloning or expression vector is not pure inthat it may comprise only a small percentage of the material in the cellin which it resides. Such a nucleic acid is isolated, however, as theterm is used herein.

Methods to deliver expression vectors or expression constructs intocells are well known to those of skill in the art. Nucleic acids,including expression vectors, can be delivered to prokaryotic andeukaryotic cells by various methods well known to those of skill in therelevant biological arts. Methods for the delivery of nucleic acids to acell in accordance to some aspects of this invention, include, but arenot limited to, different chemical, electrochemical and biologicalapproaches, for example, heat shock transformation, electroporation,transfection, for example liposome-mediated transfection,DEAE-Dextran-mediated transfection or calcium phosphate transfection. Insome embodiments, a nucleic acid construct, for example an expressionconstruct comprising a fusion protein nucleic acid sequence, isintroduced into the host cell using a vehicle, or vector, fortransferring genetic material. Vectors for transferring genetic materialto cells are well known to those of skill in the art and include, forexample, plasmids, artificial chromosomes, and viral vectors. Methodsfor the construction of nucleic acid constructs, including expressionconstructs comprising constitutive or inducible heterologous promoters,knockout and knockdown constructs, as well as methods and vectors forthe delivery of a nucleic acid or nucleic acid construct to a cell arewell known to those of skill in the art.

In one embodiment, a genetic clusters includes a nucleotide sequencethat is at least about 85% or more homologous or identical to the entirelength of a naturally occurring genetic cluster sequence, e.g., at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more of the full lengthnaturally occurring genetic cluster sequence). In some embodiments, thenucleotide sequence is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% homologous or identical to a naturally occurringgenetic cluster sequence. In some embodiments, the nucleotide sequenceis at least about 85%, e.g., is at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% homologous or identical to a geneticcluster sequence, in a fragment thereof or a region that is much moreconserved, such as an essential, but has lower sequence identity outsidethat region.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows. Todetermine the percent identity of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). The length of a reference sequencealigned for comparison purposes is at least 80% of the length of thereference sequence, and in some embodiments is at least 90% or 100%. Thenucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein nucleic acid “identity” is equivalent to nucleic acid“homology”). The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.

In many cases the nucleic acids described herein having naturallyoccurring nucleotides and are not modified. In some instances, thenucleic acids may include non-naturally occurring nucleotides and/orsubstitutions, i.e. Sugar or base substitutions or modifications.

One or more substituted sugar moieties include, e.g., one of thefollowing at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃ OCH₃, OCH₃O(CH₂)n CH₃, O(CH₂)n NH₂ or O(CH₂)n CH₃ where n is from 1 to about 10;Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl oraralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; SOCH3; SO2 CH3; ONO2; N3; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of a nucleic acid; or a groupfor improving the pharmacodynamic properties of a nucleic acid and othersubstituents having similar properties. Similar modifications may alsobe made at other positions on the nucleic acid, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Nucleic acids may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group.

Nucleic acids can also include, additionally or alternatively,nucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C) and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, isocytosine, pseudoisocytosine, as well as syntheticnucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine,2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or otherheterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine,5-bromouracil, 5-hydroxymethyluracil, 5-propynyluracil, 8-azaguanine,7-deazaguanine, N6 (6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine,2-chloro-6-aminopurine and 2,6-diaminopurine or other diaminopurines.See, e.g., Kornberg, “DNA Replication,” W. H. Freeman & Co., SanFrancisco, 1980, pp 75-T7; and Gebeyehu, G., et al. Nucl. Acids Res.,15:4513 (1987)). A “universal” base known in the art, e.g., inosine, canalso be included.

In the context of the present disclosure, hybridization means basestacking and hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases. For example, adenine and thymine are complementarynucleobases which pair through the formation of hydrogen bonds.Complementary, as the term is used in the art, refers to the capacityfor precise pairing between two nucleotides. For example, if anucleotide at a certain position of an nucleic acid is capable ofhydrogen bonding with a nucleotide at the same position of a secondnucleic acid, then the two nucleic acids are considered to becomplementary to each other at that position. The nucleic acids arecomplementary to each other when a sufficient number of correspondingpositions in each molecule are occupied by nucleotides that can hydrogenbond with each other through their bases. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity or precise pairing such that stableand specific binding occurs between the nucleic acids. 100%complementarity is not required.

Various aspects of the embodiments described above may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

As shown in the examples, the refactoring approach has been applied tothe nif gene cluster from Klebsiella oxytoca encoding the nitrogenfixation pathway for converting atmospheric N2 to ammonia. The nativegene cluster consists of 20 genes in seven operons and is encoded in23.5 kb of DNA. The refactored gene cluster may share little DNAsequence identity with the wild type (WT).

When the nif cluster is a native nif cluster, it may have the DNAsequence of any naturally occurring nif cluster. For example it may havethe sequence of a naturally occurring nif cluster from Klebsiellaoxytoca (SEQ ID NO. 4) Pseudomonas stutzi nif cluster (SEQ ID NO. 5) andPaenibacillus nif cluster. Refactored nif clusters may be any refactorednif cluster which is active in producing the proteins involved inpromoting N₂ conversion to other nitrogen forms.

The following exemplary DNA sequences of nif clusters are usefulaccording to the invention.

refactored nif cluster v1.0 (SEQ ID NO. 1)taatacgactcactatagggagaacaataaactaacataaggaggataaatatgaccatgcgtcagtgcgcgatttatggcaaaggtggtattggcaaaagcacgacgacccagaacttggtggcggccctggccgagatgggtaaaaaggttatgattgtgggttgcgacccgaaggccgacagcacgcgcctgattctgcacgcgaaagcacaaaacacgattatggagatggctgccgaggttggtagcgtggaggatctggagctggaggacgttctgcaaattggttacggtgatgttcgttgcgcagagagcggtggtccggaaccaggtgtcggctgtgcgggtcgtggtgtaattaccgctatcaatttcctggaagaagagggtgcgtacgaagatgatctggatttcgttttctacgatgtgctgggtgatgtcgtgtgcggtggttttgcaatgccgattcgcgagaataaggcacaagaaatttacattgtctgtagcggcgagatgatggcaatgtacgctgctaacaacatcagcaagggtattgttaaatacgcaaaaagcggtaaggttcgcttgggtggtttgatttgcaacagccgtcagaccgaccgtgaggacgaactgatcatcgccctggctgagaaactgggcacccaaatgatccacttcgtgccacgcgataatattgttcaacgtgcagaaatccgccgtatgaccgtcattgagtatgacccggcatgcaagcaagcgaacgagtaccgcaccttggcacagaaaatcgtgaacaacaccatgaaggttgttccgacgccgtgtacgatggacgagctggagagcctgctgatggagttcggcattatggaggaggaggacaccagcattatcggtaagaccgcagcggaggagaatgcggcataagcgtgcgtacaccttaatcaccgcttcatgctaaggtcctggctgcatgcaaaaattcacatccctatctagcggaggagccggatgatgactaatgctactggcgaacgtaacctggcactgattcaagaagtactggaagtgttcccggaaaccgcgcgcaaagagcgccgtaaacacatgatggtttctgacccggaaatggaatctgtgggtaaatgcatcatctctaatcgcaaatctcagccgggtgtcatgactgttcgtggctgtgcgtacgcaggttctaaaggtgtcgtattcggcccgatcaaagatatggcgcatatctctcatggcccggtaggctgtggccagtactctcgcgcgggacgtcgtaactactacacgggcgtttctggcgttgactctttcggcacgctgaacttcacctctgacttccaggaacgtgacatcgttttcggtggcgataaaaagctgtccaaactgatcgaagaaatggaactgctgttcccgctgactaaaggcattactatccaaagcgaatgtccggtgggtctgatcggtgatgacatcagcgcggtcgcaaacgcatcttccaaagccctggataagccggtgatcccggttcgttgcgagggcttccgcggcgtttctcagtctctgggtcatcacatcgcaaacgatgttgtgcgtgactggattctgaacaaccgtgaaggtcagccttttgaaaccaccccttatgacgttgcgattattggcgactataacatcggcggcgacgcctgggcatcccgcatcctgctggaggagatgggtctgcgtgttgtcgcacagtggtctggcgatggcaccctggttgaaatggaaaacaccccgtttgttaaactgaacctggttcactgctaccgctccatgaactacattgcccgtcacatggaagaaaaacatcagatcccttggatggaatacaacttcttcggtccgactaaaatcgcagaatccctgcgtaaaatcgccgatcagtttgatgataccattcgcgcgaacgctgaagcagtaattgcgcgctacgaaggccagatggcagcaatcattgctaagtaccgtccgcgcctggaaggtcgtaaagtgctgctgtacatgggtggtctgcgtccacgtcatgtgatcggtgcctacgaggacctgggcatggagatcatcgcagcgggttacgaatttgcacacaacgacgactatgatcgtacgctgccagacctgaaagaaggtacgctgctgtttgacgacgccagctcttatgaactggaagccttcgtgaaagcgctgaaaccagacctgatcggctccggcatcaaggaaaaatacattttccagaaaatgggcgtgccgttccgccagatgcactcctgggactactccggtccgtaccacggctacgacggtttcgctatcttcgctcgtgacatggatatgaccctgaataacccagcgtggaatgaactgaccgcaccgtggctgaaatctgcataacaaacaccccatgtcgatactgaacgaatcgacgcacactcccttccttgcaatctcatactctcaaaaattaggcgaggtaacatgtctcaaactatcgataaaatcaactcttgttacccgctgttcgagcaggacgaatatcaggaactgttccgtaacaaacgtcagctggaagaagcgcacgacgcacagcgcgtgcaggaagtgttcgcatggaccaccaccgcggaatacgaagctctgaacttccagcgcgaagccctgacggttgatccggcgaaagcgtgccagcctctgggtgcggttctgtgcagcctgggttttgccaacaccctgccgtatgtccacggttcccagggctgcgtagcctacttccgtacctatttcaaccgccactttaaagaaccaatcgcgtgcgtgtccgacagcatgacggaggacgcggcagttttcggtggtaacaacaacatgaacctgggcctgcaaaatgcttccgcactgtacaaaccggaaatcatcgcagtgtctaccacctgcatggcagaggttattggtgatgatctgcaagcatttattgccaacgcaaagaaagacggtttcgttgacagctctatcgcggttccgcacgctcataccccgtccttcatcggttctcacgtaactggttgggacaacatgttcgaaggcttcgcaaaaacttttaccgcagactatcaaggccaaccgggtaaactgccgaagctgaacctggtgaccggctttgaaacctacctgggcaactttcgtgtcctgaagcgcatgatggagcagatggcggttccgtgttctctgctgtctgacccgtctgaggttctggacactccagcggacggccactatcgcatgtattctggtggcaccactcagcaggaaatgaaagaggccccagacgcgattgacaccctgctgctgcaaccgtggcagctgctgaaaagcaagaaagttgttcaggaaatgtggaaccagccggcaacggaagttgcaatcccgctgggtctggcagctactgacgaactgctgatgaccgtgtcccaactgagcggcaaaccaatcgcggatgctctgaccctggaacgcggtcgcctggtggacatgatgctggacagccacacgtggctgcatggcaagaaatttggcctgtacggtgacccggacttcgtaatgggcctgacccgtttcctgctggaactgggctgcgagccgactgttatcctgtctcacaacgctaacaaacgttggcagaaggccatgaacaaaatgctggatgcgagcccatacggccgtgatagcgaagtgttcatcaactgcgacctgtggcatttccgctctctgatgtttacgcgtcagccggatttcatgatcggtaactcttacggcaaattcatccagcgtgacactctggccaaaggcaaagcgtttgaagtgccgctgattcgtctgggctttccgctgttcgaccgtcaccacctgcaccgccagaccacctggggttacgaaggcgcgatgaacatcgtaactactctggtaaacgcagtactggaaaagctggacagcgatacttcccagctgggcaaaaccgactattctttcgatctggttcgttaacctgattgtatccgcatctgatgctaccgtggttgagttaccatactcactcccggaggtacttctatgtctgacaatgataccctgttttggcgcatgctggcgctgtttcagtcgctgccggatttgcagccggctcaaatcgtcgattggctggcgcaggaatccggcgaaaccctgacgccggagcgccttgccaccctgacccaaccgcaactcgcggcgtcgttcccatccgcgacggcagtgatgagcccggctcgctggagccgcgttatggcttctctgcaaggcgccctcccagcccacttgcgcatcgtacgtccggcgcagcgtaccccgcaactgctcgccgcgttttgcagccaagacggccttgttatcaatggtcatttcggccagggtcgtctgttcttcatttacgcctttgacgagcagggcggctggctgtatgacttgcgccgctatccgagcgcaccgcaccagcaggaagcgaatgaggtgcgtgctcgtctgattgaagattgccagctgctgttctgccaggagattggcggtccggcagcagcgcgtctgatccgccaccgcatccatccgatgaaggcgcagccgggtactacgattcaggcgcagtgtgaagctatcaacaccctgctggccggtcgcctgccgccgtggctcgccaaacgtttgaaccgtgataacccgctggaagagcgtgtgttttaacatttttgccttgcgacagacctcctacttagattgccacactattcaatacatcactggaggttattacaaatgaagggtaacgagattcttgctctgctggacgaaccggcctgtgaacacaaccataaacagaaatcc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aattgatggagcgtaccggctgctatatggaagttgttgccgcctaacattgtaatagccaccaaaagagtgatgatagtcatgggtgatacccgtagaccattctgaaatcgaaggaggttttccatgaaacaagtgtacctggacaacaacgcgaccacccgcctggacccgatggttctggaagcgatgatgccgtttctcacggatttctatggcaatccgtccagcatccatgacttcggcatcccggcacaagcggcgctggaacgtgcgcaccagcaagctgcggcactgctgggcgcagagtacccgtctgaaatcattttcacgagctgtgcgaccgaggccactgcaaccgccattgcgtcggccatcgcgttattgccggaacgccgcgaaatcatcacctcggtagtggagcacccggctacgctggcggcgtgcgagcacctggaacgccaaggctatcgcatccatcgcattgcggtggatagcgaaggtgcgctggacatggcccagttccgtgcagcgctctcgccgcgtgtcgcgttggtgagcgtgatgtgggccaacaacgaaaccggcgtgctgttcccgattggcgaaatggccgagcttgcccacgagcagggcgctctgttccactgcgatgccgttcaggtcgttggcaaaatcccaattgctgttggccagacgcgcatcgacatgctgtcttgctccgcgcacaagtttcatggtccgaagggtgttggttgcttgtacttacgtcgtggcacgcgctttcgtccgctgcttcgcggtggccatcaagaatatggtcgccgtgccggcactgagaatatctgtggcatcgtcggcatgggcgctgcgtgcgaactggcgaacatccatctgccgggtatgacccatattggccagttacgcaatcgcctggagcaccatctactcgccagcgtaccgtccgtgatggttatgggcggtagtcagccgcgtgtaccgggtactgtcaacctggcgttcgagtttatcgaaggtgaagcgatcctgctcttgctgaaccaggctggcattgccgcaagctccggctccgcgtgtacctctggcagcttggagccgagccatgtgatgcgcgccatgaacattccatacaccgcggctcacggcaccattcgttttagcctgagccgttatacgcgcgagaaagagatcgactacgtcgttgcgaccctcccgccaatcattgatcgtctgcgtgccttgtccccgtattggcagaatggtaagccgcgtccggcagatgcagtctttaccccggtttacggttaagagttactggccctgatttctccgcttctaataccgcacagcgactaggagcctaactcgccacaaggaaacatatggagcgcgtcttgatcaacgatactaccctgcgtgatggcgaacaatctccgggcgtagcgtttcgtacctccgagaaagttgccatcgcggaggcactgtacgctgcgggtatcaccgcgatggaagtcggcactccggcgatgggtgatgaagagatcgcccgcattcagctggtgcgtcgtcaactgccggacgcgacgcttatgacctggtgccgtatgaacgctctggaaatccgtcagagcgcggatctgggtattgactgggtggatatctcgatcccagcatccgacaagctgcgtcagtacaagctgcgtgagccgctggccgtgctgctggagcgccttgcgatgtttatccatctggcccacacgttaggcctcaaagtatgtattggttgcgaggatgcgagccgtgcgtctggtcagaccctgcgcgccattgccgaggtggcccagcaatgcgcggctgcgcgcttgcgttacgctgacaccgtgggcctgctggacccgttcaccaccgcagcccagatcagcgccctgcgtgacgtttggtcgggcgagatcgagatgcatgctcacaatgatctgggcatggctaccgcgaacacgctggcggcagtttcggctggcgccacgtcggtgaacactaccgtcctcggtctgggtgaacgtgcaggcaacgcagccctggaaaccgttgcgctgggcctggaacgctgcctgggcgtggaaaccggcgtccatttcagcgcgctcccagcgctctgtcagcgcgtcgcggaggctgcacagcgcgcaatcgacccgcaacagccgctggtgggtgaattggttttcacccacgagtctggtgttcacgttgcggcgctgctgcgcgacagcgaatcctatcaatctattgccccaagcctcatgggccgtagctaccgtctggtgctcggcaagcattcgggtcgtcaggctgtcaacggtgttttcgaccagatgggttaccacctgaatgcggcgcagatcaatcagttgctgccggccattcgccgcttcgccgagaattggaaacgctctccgaaagactacgaactggttgcgatctatgacgaattgtgcggtgaatccgcccttcgtgctcgcggctaagactcaacacgctagggacgtgaagtcgattccttcgatgcagaaggcgagaactagatttaagggccattatagatggagtggttttaccagattccgggtgtagacgaattgcgcagcgctgaatccttctttcagttcttcgcggttccataccagccggaactgctgggccgctgctcgcttccggtgttagcgacgttccaccgtaaactgcgtgcggaggtcccgctgcaaaaccgtctggaggacaatgatcgtgcgccgtggctcttggcgcgccgcctcctggccgaatcttatcagcagcaatttcaggagagcggcacctaatcgagaaacaaggcagttccgggctgaaagtagcgccgggacaagtcccgtattataaccgcctaggaggtgttggatgcgcccgaaattcaccttctctgaagaggtccgcgtagttcgcgcgattcgtaatgatggcaccgtggcgggttttgcgccaggtgcgctgctggttcgtcgcggttcgacgggctttgtgcgtgactggggtgtgttcctgcaagaccagatcatctatcaaatccactttccggaaaccgaccgcattatcggctgtcgcgagcaggagttaatcccgattacccagccgtggttggctggtaacctccagtatcgtgacagcgtcacgtgccaaatggcactggctgtcaacggtgacgtggttgtgagcgccggtcaacgtggccgtgtggaggccactgatcgtggcgaacttggcgattcctacaccgtggacttcagcggccgttggttccgcgttccggtccaggccatcgcgctgattgaagagcgcgaagaataaacgccacgcgtagtgagacatacacgttcgttgggttcactcagagactgaagttattacccaggaggtctataatgaatccgtggcagcgctttgcccgtcaacgccttgctcgcagccgctggaaccgtgatccggctgctctcgacccagccgataccccagcgttcgagcaggcgtggcagcgtcaatgccatatggaacaaaccatcgtagcgcgtgtcccggaaggcgatattccggctgccttactggaaaacatcgcggccagcctggcgatctggctggacgagggtgacttcgctccgccggagcgcgctgcgattgtgcgtcatcatgcacgtctggagctggcgtttgccgacattgcccgccaggcaccgcaaccggatctgagcacggttcaagcgtggtatctgcgtcaccagactcaattcatgcgtccggagcagcgtctgacccgtcacctgctcctgacggtcgataatgatcgcgaggcggtgcatcaacgcatccttggcctgtatcgtcagatcaacgcgagccgtgacgccttcgccccactggcacagcgccactctcattgcccgtccgccttggaagaaggccgtctgggctggatctcccgtggtctgctgtacccgcagctcgaaaccgcgttgtttagcctggcggaaaacgcactgtcgctgccgattgcgtcggaattgggttggcacctgttatggtgcgaggccattcgtccggcagccccgatggagccgcaacaggcccttgaatctgcgcgcgactacttgtggcagcagagccagcagcgccaccagcgtcaatggctggagcagatgatttcccgccaaccgggcctgtgtggttaatagcataaccccttggggcctctaaacgggtcttgaggggttttttgt refactored nif cluster v2.1(SEQ ID NO. 2)taatacgactcactattgggagatACAAATATATAATATATTTAAGGAGGTTTCATATATGACCATGCGTCAGTGCGCGATTTATGGCAAAGGTGGTATTGGCAAAAGCACGACGACCCAGAACTTGGTGGCGGCCCTGGCCGAGATGGGTAAAAAGGTTATGATTGTGGGTTGCGACCCGAAGGCCGACAGCACGCGCCTGATTCTGCACGCGAAAGCACAAAACACGATTATGGAGATGGCTGCCGAGGTTGGTAGCGTGGAGGATCTGGAGCTGGAGGACGTTCTGCAAATTGGTTACGGTGATGTTCGTTGCGCAGAGAGCGGTGGTCCGGAACCAGGTGTCGGCTGTGCGGGTCGTGGTGTAATTACCGCTATCAATTTCCTGGAAGAAGAGGGTGCGTACGAAGATGATCTGGATTTCGTTTTCTACGATGTGCTGGGTGATGTCGTGTGCGGTGGTTTTGCAATGCCGATTCGCGAGAATAAGGCACAAGAAATTTACATTGTCTGTAGCGGCGAGATGATGGCAATGTACGCTGCTAACAACATCAGCAAGGGTATTGTTAAATACGCAAAAAGCGGTAAGGTTCGCTTGGGTGGTTTGATTTGCAACAGCCGTCAGACCGACCGTGAGGACGAACTGATCATCGCCCTGGCTGAGAAACTGGGCACCCAAATGATCCACTTCGTGCCACGCGATAATATTGTTCAACGTGCAGAAATCCGCCGTATGACCGTCATTGAGTATGACCCGGCATGCAAGCAAGCGAACGAGTACCGCACCTTGGCACAGAAAATCGTGAACAACACCATGAAGGTTGTTCCGACGCCGTGTACGATGGACGAGCTGGAGAGCCTGCTGATGGAGTTCGGCATTATGGAGGAGGAGGACACCAGCATTATCGGTAAGACCGCAGCGGAGGAGAATGCGGCATAATACTCGAACCCCTAGCCCGCTCTTATCGGGCGGCTAGGGGTTTTTTGTCGAAGAACAGATATGAAAGTGTTAGAACTGTAATACGACTCACTATAGGTAGAGCGTGCGTACACCTTAATCACCGCTTCATGCTAAGGTCCTGGCTGCATGCAAAAATTCACATTTTTATCTAGCGGAGGAGCCGGatgatgactaatgctactggcgaacgtaacctggcactgattcaagaagtactggaagtgttcccggaaaccgcgcgcaaagagcgccgtaaacacatgatggtttctgacccgGaaatgGaatctgtgggtaaatgcatcatctctaatcgcaaatctcagccgggtgtcatgactgttcgtggctgtgcgtacgcaggttctaaaggtgtcgtattcggcccgatcaaagatatggcgcatatctctcatggcccggTaggctgtggccagtactctcgcgcggGacgtcgtaactactacacgggcgtttctggcgttgactctttcggcacgctgaacttcacctctgacttccaggaacgtgacatcgttttcggtggcgataaaaagctgtccaaactgatcgaagaaatggaactgctgttcccgctgactaaaggcattactatccaaagcgaatgtccggtgggtctgatcggtgatgacatcagcgcggtcgcaaacgcatcttccaaagccctggataagccggtgatcccggttcgttgcgagggcttccgcggcgtttctcagtctctgggtcatcacatcgcaaacgatgttgtgcgtgactggattctgaacaaccgtgaaggtcagccttttgaaaccaccccttatgacgttgcgattattggcgactataacatcggcggcgacgcctgggcatcccgcatcctgctggaggagatgggtctgcgtgttgtcgcacagtggtctggcgatggcaccctggttgaaatggaaaacaccccgtttgttaaactgaacctggttcactgctaccgctccatgaactacattgcccgtcacatggaagaaaaacatcagatcccttggatggaatacaacttcttcggtccgactaaaatcgcagaatccctgcgtaaaatcgccgatcagtttgatgataccattcgcgcgaacgctgaagcagtaattgcgcgctacgaaggccagatggcagcaatcattgctaagtaccgtccgcgcctggaaggtcgtaaagtgctgctgtacatgggtggtctgcgtccacgtcatgtgatcggtgcctacgaggacctgggcatggagatcatcgcagcgggttacgaatttgcacacaacgacgactatgatcgtacgctgccagacctgaaagaaggtacgctgctgtttgacgacgccagctcttatgaactggaagccttcgtgaaagcgctgaaaccagacctgatcggctccggcatcaaggaaaaatacattttccagaaaatgggcgtgccgttccgccagatgcactcctgggactactccggtccgtaccacggctacgacggtttcgctatcttcgctcgtgacatggatatgaccctgaataacccagcgtggaatgaactgaccgcaccgtggctgaaatctgcataaCAAACACCCCATGTCGATACTGAACGAATCGACGCACACTCCCTTCCTTGCAATCTCATACTCTCAAAAATTAGGCGAGGTAACatgtctcaaactatcgataaaatcaactcttgttacccgctgttcgagcaggacgaatatcaggaactgttccgtaacaaacgtcagctggaagaagcgcacgacgcacagcgcgtgcaggaagtgttcgcatggaccaccaccgcggaatacgaagctctgaacttccagcgcgaagccctgacggttgatccggcgaaagcgtgccagcctctgggtgcggttctgtgcagcctgggttttgccaacaccctgccgtatgtccacggttcccagggctgcgtagcctacttccgtacctatttcaaccgccactttaaagaaccaatcgcgtgcgtgtccgacagcatgacggaggacgcggcagttttcggtggtaacaacaacatgaacctgggcctgcaaaatgcttccgcactgtacaaaccggaaatcatcgcagtgtctaccacctgcatggcagaggttattggtgatgatctgcaagcatttattgccaacgcaaagaaagacggtttcgttgacagctctatcgcggttccgcacgctcataccccgtccttcatcggttctcacgtaactggttgggacaacatgttcgaaggcttcgcaaaaacttttaccgcagactatcaaggccaaccgggtaaactgccgaagctgaacctggtgaccggctttgaaacctacctgggcaactttcgtgtcctgaagcgcatgatggagcagatggcggttccgtgttctctgctgtctgacccgtctgaggttctggacactccagcggacggccactatcgcatgtattctggtggcaccactcagcaggaaatgaaagaggccccagacgcgattgacaccctgctgctgcaaccgtggcagctgctgaaaagcaagaaagttgttcaggaaatgtggaaccagccggcaacggaagttgcaatcccgctgggtctggcagctactgacgaactgctgatgaccgtgtcccaactgagcggcaaaccaatcgcggatgctctgaccctggaacgcggtcgcctggtggacatgatgctggacagccacacgtggctgcatggcaagaaatttggcctgtacggtgacccggacttcgtaatgggcctgacccgtttcctgctggaactgggctgcgagccgactgttatcctgtctcacaacgctaacaaacgttggcagaaggccatgaacaaaatgctggatgcgagcccatacggccgtgatagcgaagtgttcatcaactgcgacctgtggcatttccgctctctgatgtttacgcgtcagccggatttcatgatcggtaactcttacggcaaattcatccagcgtgacactctggccaaaggcaaagcgtttgaagtgccgctgattcgtctgggctttccgctgttcgaccgtcaccacctgcaccgccagaccacctggggttacgaaggcgcgatgaacatcgtaactactctggtaaacgcagtactggaaaagctggacagcgatacttcccagctgggcaaaaccgactattctttcgatctggttcgttaaCCTGATTGTATCCGCATCTGATGCTACCGTGGTTGAGTTACCATACTCACTCCCGGAGGTACTTCTATGTCTGACAATGATACCCTGTTTTGGCGCATGCTGGCGCTGTTTCAGTCGCTGCCGGATTTGCAGCCGGCTCAAATCGTCGATTGGCTGGCGCAGGAATCCGGCGAAACCCTGACGCCGGAGCGCCTTGCCACCCTGACCCAACCGCAACTCGCGGCGTCGTTCCCATCCGCGACGGCAGTGATGAGCCCGGCTCGCTGGAGCCGCGTTATGGCTTCTCTGCAAGGCGCCCTCCCAGCCCACTTGCGCATCGTACGTCCGGCGCAGCGTACCCCGCAACTGCTCGCCGCGTTTTGCAGCCAAGACGGCCTTGTTATCAATGGTCATTTCGGCCAGGGTCGTCTGTTCTTCATTTACGCCTTTGACGAGCAGGGCGGCTGGCTGTATGACTTGCGCCGCTATCCGAGCGCACCGCACCAGCAGGAAGCGAATGAGGTGCGTGCTCGTCTGATTGAAGATTGCCAGCTGCTGTTCTGCCAGGAGATTGGCGGTCCGGCAGCAGCGCGTCTGATCCGCCACCGCATCCATCCGATGAAGGCGCAGCCGGGTACTACGATTCAGGCGCAGTGTGAAGCTATCAACACCCTGCTGGCCGGTCGCCTGCCGCCGTGGCTCGCCAAACGTTTGAACCGTGATAACCCGCTGGAAGAGCGTGTGTTTTAACATTTTTGCCTTGCGACAGACCTCCTACTTAGATTGCCACACTATTCAATTCATCACTGGAGGTTATTACAAATGAAGGGTAACGAGATTCTTGCTCTGCTGGACGAACCGGCCTGTGAACACAACCATAAACAGAAATCCGGCTGTAGCGCCCCAAAGCCGGGTGCGACGGCGGCTGGCTGCGCTTTCGATGGTGCGCAGATCACCCTGCTCCCGATTGCGGACGTTGCCCACCTCGTGCATGGCCCAATCGGTTGCGCAGGTAGCTCTTGGGACAACCGTGGCAGCGCCTCCAGCGGTCCGACCCTGAATCGTTTGGGCTTTACCACTGACTTGAATGAACAAGATGTGATCATGGGTCGCGGCGAGCGTCGCCTGTTCCACGCTGTGCGCCATATTGTCACCCGTTACCACCCAGCGGCAGTATTCATCTACAATACGTGCGTGCCGGCTATGGAAGGCGATGACCTGGAGGCCGTGTGTCAGGCAGCCCAGACTGCGACCGGCGTCCCGGTAATCGCAATTGATGCGGCTGGCTTCTACGGTTCGAAGAACCTGGGCAACCGTCCGGCAGGCGATGTCATGGTTAAACGCGTCATTGGCCAACGTGAGCCAGCGCCGTGGCCGGAGAGCACCCTGTTTGCCCCGGAGCAACGTCATGACATTGGCTTGATCGGTGAGTTCAACATTGCGGGCGAGTTTTGGCACATTCAGCCGCTGCTTGATGAGCTGGGTATCCGCGTTTTGGGTTCGCTCAGCGGCGATGGTCGTTTCGCCGAGATTCAAACCATGCACCGTGCCCAGGCGAACATGCTGGTGTGCAGCCGTGCTCTGATCAATGTTGCGCGTGCTCTGGAACAGCGCTATGGCACCCCGTGGTTTGAAGGCTCGTTCTATGGTATCCGCGCGACCAGCGACGCCCTGCGCCAGTTAGCGGCGCTGCTGGGCGATGACGACCTCCGTCAGCGCACCGAGGCGCTGATCGCGCGTGAAGAACAGGCGGCTGAGCTGGCCCTGCAACCGTGGCGTGAACAGCTGCGTGGCCGCAAGGCCCTGCTCTACACGGGTGGTGTCAAAAGCTGGTCTGTGGTGTCCGCGCTTCAGGATCTGGGTATGACCGTGGTTGCCACGGGCACGCGTAAGAGCACGGAAGAGGATAAACAGCGCATCCGCGAATTGATGGGCGAAGAGGCCGTGATGCTTGAAGAAGGCAACGCACGTACCTTATTGGATGTAGTTTATCGCTATCAAGCAGACCTGATGATTGCCGGTGGCCGCAACATGTATACCGCCTACAAAGCGCGCTTGCCGTTCCTGGACATCAACCAGGAACGCGAGCACGCGTTTGCGGGCTACCAAGGCATCGTGACCTTAGCGCGCCAGCTGTGCCAAACGATTAACAGCCCGATCTGGCCGCAGACTCATTCCCGCGCACCGTGGCGCTAATGTCACGCTAGGAGGCAATTCTATAAGAATGCACACTGCACCTAAACCTACCACACCTGGAAGAAGTAATTATGGCAGACATTTTCCGCACTGATAAGCCGTTGGCTGTGTCGCCGATCAAGACCGGCCAGCCGCTGGGTGCGATCCTGGCGTCCCTGGGTATCGAGCACTCGATTCCGCTGGTACATGGCGCGCAGGGCTGTTCGGCTTTTGCCAAGGTTTTCTTTATCCAGCACTTCCACGATCCGGTCCCGCTGCAAAGCACGGCAATGGACCCGACCAGCACCATCATGGGCGCTGATGGTAACATCTTCACCGCGCTGGACACTCTCTGCCAACGCAATAACCCGCAAGCAATTGTGCTGCTGAGCACCGGCCTCTCCGAGGCGCAGGGCAGCGACATTTCCCGTGTAGTGCGTCAGTTCCGTGAAGAATATCCGCGTCATAAAGGCGTGGCGATTCTGACTGTTAACACCCCGGACTTTTACGGTAGCATGGAGAACGGCTTTTCCGCTGTCCTGGAGTCTGTGATTGAACAGTGGGTTCCGCCAGCCCCACGTCCGGCGCAGCGCAATCGTCGCGTCAATCTTTTGGTGAGCCATCTCTGTAGCCCAGGCGATATTGAGTGGCTGCGCCGTTGCGTCGAGGCCTTCGGTCTGCAACCGATCATTCTGCCGGATCTGGCTCAGAGCATGGACGGCCACCTTGCTCAGGGTGACTTTTCGCCGCTGACGCAGGGCGGCACGCCGTTGCGCCAAATCGAGCAGATGGGCCAGAGCCTTTGCTCTTTTGCGATTGGCGTCAGCCTGCACCGTGCGAGCAGCCTGCTGGCTCCGCGTTGTCGTGGCGAAGTCATCGCCTTGCCGCACCTCATGACCTTGGAACGCTGCGACGCCTTTATCCATCAGTTGGCGAAAATCAGCGGTCGCGCCGTTCCGGAGTGGCTGGAACGCCAGCGCGGTCAGCTGCAAGACGCCATGATCGATTGCCACATGTGGCTGCAAGGCCAGCGCATGGCGATTGCCGCCGAAGGCGACCTGCTGGCAGCGTGGTGCGATTTCGCGAACTCTCAAGGTATGCAGCCGGGTCCACTGGTTGCTCCGACGGGTCATCCGAGCCTGCGTCAGTTGCCGGTGGAGCGCGTGGTGCCGGGTGATCTGGAGGATCTTCAGACCCTCTTATGCGCACATCCGGCCGACTTACTGGTGGCGAACTCCCACGCCCGTGATTTAGCAGAGCAATTCGCCCTGCCGCTGGTGCGCGCAGGCTTCCCGCTGTTTGACAAACTGGGCGAATTTCGTCGTGTTCGCCAGGGTTATAGCGGTATGCGTGATACCCTGTTCGAGTTGGCGAACCTGATCCGTGAACGCCATCATCATCTGGCTCATTATCGCAGCCCGCTGCGCCAGAACCCAGAATCCTCGTTGTCTACGGGTGGCGCGTACGCAGCGGATTAActagagattaaTATggagaaattaagcATGAAAACTATGGACGGTAACGCTGCGGCTGCATGGATTAGCTACGCCTTTACCGAAGTGGCTGCGATCTACCCGATTACGCCGAGCACCCCGATGGCGGAAAATGTGGACGAATGGGCTGCGCAGGGCAAGAAGAACCTCTTCGGCCAGCCGGTGCGCCTGATGGAGATGCAGTCGGAAGCGGGTGCAGCAGGTGCTGTGCATGGCGCCTTGCAAGCTGGCGCACTGACGACCACCTACACCGCGTCGCAGGGCCTGTTGCTGATGATCCCAAACATGTACAAAATCGCGGGTGAACTGCTGCCGGGTGTCTTTCATGTTTCGGCACGCGCACTGGCCACCAATAGCCTCAACATCTTTGGCGATCATCAGGATGTAATGGCGGTGCGCCAAACGGGCTGCGCGATGTTGGCCGAGAATAACGTCCAGCAAGTTATGGATTTGTCCGCGGTAGCCCACTTGGCAGCGATCAAAGGTCGCATTCCGTTCGTGAACTTCTTCGATGGCTTTCGCACCAGCCACGAAATCCAGAAGATCGAGGTTCTGGAATATGAACAGCTGGCCACCTTGTTGGATCGTCCGGCCCTGGACAGCTTCCGCCGTAACGCCCTTCACCCGGACCACCCGGTCATCCGTGGCACCGCCCAGAACCCGGACATCTACTTCCAGGAACGTGAGGCCGGTAACCGTTTCTATCAGGCGCTCCCGGATATTGTGGAATCTTACATGACCCAGATTTCTGCCCTGACTGGTCGCGAGTATCACCTGTTTAACTACACTGGTGCTGCGGATGCGGAGCGCGTGATCATCGCGATGGGCTCTGTCTGTGACACCGTCCAAGAGGTGGTTGACACGCTGAATGCAGCGGGTGAGAAAGTTGGTCTGCTCTCCGTTCATCTTTTCCGCCCGTTTTCGTTAGCGCACTTCTTCGCCCAACTGCCGAAAACTGTACAGCGTATCGCAGTATTGGACCGTACGAAAGAGCCAGGTGCTCAAGCAGAGCCGCTGTGCCTCGATGTGAAGAATGCCTTTTACCACCATGACGATGCCCCGTTGATTGTGGGTGGTCGCTATGCCTTGGGCGGTAAGGACGTGTTGCCGAACGATATTGCGGCCGTGTTTGATAACCTGAACAAACCGCTGCCGATGGACGGCTTCACGCTGGGTATCGTGGACGATGTTACCTTCACCTCTCTCCCGCCAGCGCAGCAGACCCTGGCGGTTTCTCACGACGGCATCACGGCATGTAAGTTTTGGGGCATGGGCTCCGACGGCACGGTTGGTGCGAACAAGTCCGCGATCAAGATTATCGGCGACAAAACGCCACTGTATGCGCAAGCGTACTTTTCCTACGACTCGAAGAAGAGCGGTGGTATTACCGTCAGCCATCTGCGTTTTGGTGATCGCCCGATCAACTCCCCGTATTTGATCCATCGCGCGGATTTCATCTCGTGCAGCCAGCAAAGCTATGTTGAACGCTACGATCTGCTGGATGGCCTTAAACCGGGTGGCACCTTTCTGCTGAACTGCTCCTGGAGCGATGCCGAACTGGAGCAACATCTGCCGGTCGGTTTCAAACGTTATCTGGCACGCGAGAATATCCACTTCTACACTCTCAACGCTGTGGACATCGCCCGTGAGCTTGGTTTGGGTGGCCGTTTCAACATGCTGATGCAGGCTGCCTTCTTCAAACTGGCCGCGATCATTGACCCGCAGACTGCTGCGGACTATCTGAAGCAGGCTGTTGAGAAAAGCTATGGCAGCAAAGGTGCGGCGGTCATCGAGATGAACCAGCGTGCCATCGAGCTTGGCATGGCCAGCCTGCACCAGGTGACGATCCCGGCACATTGGGCCACCCTGGATGAGCCAGCGGCGCAGGCGTCCGCGATGATGCCGGACTTTATCCGCGACATCCTGCAACCGATGAACCGTCAGTGCGGCGACCAGCTTCCGGTGTCGGCTTTTGTCGGCATGGAAGATGGCACCTTCCCGTCCGGCACGGCCGCATGGGAGAAACGTGGCATCGCCCTTGAGGTGCCAGTCTGGCAGCCGGAAGGCTGCACGCAGTGCAACCAGTGCGCCTTCATTTGTCCGCACGCCGCGATTCGTCCGGCGTTGTTGAATGGCGAAGAGCATGATGCTGCCCCGGTTGGCCTGCTGAGCAAACCGGCACAAGGCGCTAAAGAATATCACTATCATCTGGCGATTAGCCCGCTGGACTGCTCCGGCTGTGGCAACTGCGTTGACATTTGTCCAGCTCGTGGCAAAGCGTTGAAGATGCAGTCTCTGGATAGCCAACGCCAGATGGCTCCGGTGTGGGATTATGCGCTGGCGCTGACCCCGAAGTCTAACCCGTTTCGTAAAACCACCGTCAAAGGCTCGCAGTTCGAAACCCCGCTGCTGGAGTTTAGCGGTGCGTGCGCTGGTTGTGGCGAAACGCCGTATGCGCGCCTCATTACCCAGCTGTTTGGCGACCGCATGCTGATTGCCAATGCCACCGGCTGTTCCAGCATCTGGGGCGCATCTGCGCCGAGCATCCCGTATACCACCAATCATCGTGGTCATGGTCCGGCCTGGGCGAATAGCCTGTTTGAGGACAATGCCGAATTTGGTTTAGGTATGATGCTGGGCGGTCAAGCTGTGCGTCAACAGATCGCGGACGATATGACGGCTGCGTTAGCGCTCCCGGTTTCCGATGAGCTGAGCGACGCGATGCGCCAGTGGTTGGCGAAACAGGACGAGGGTGAAGGCACGCGTGAGCGTGCGGACCGTCTGAGCGAGCGCTTAGCCGCGGAGAAAGAGGGCGTTCCGCTGTTAGAGCAGCTGTGGCAAAATCGTGATTACTTTGTGCGTCGCAGCCAGTGGATTTTCGGCGGTGACGGCTGGGCCTATGATATTGGCTTCGGTGGCCTGGACCACGTCCTCGCCAGCGGTGAGGATGTGAACATTCTGGTATTTGACACCGAAGTCTACTCGAACACCGGCGGTCAAAGCAGCAAATCGACCCCGGTCGCGGCCATCGCCAAGTTCGCGGCTCAGGGCAAGCGCACCCGCAAGAAAGACCTGGGTATGATGGCGATGAGCTACGGCAACGTCTATGTAGCCCAGGTGGCGATGGGTGCGGATAAAGATCAAACTCTGCGCGCCATTGCGGAAGCTGAAGCGTGGCCAGGCCCGTCGCTGGTGATTGCGTATGCGGCCTGCATCAATCATGGCCTGAAGGCCGGTATGCGTTGCAGCCAACGTGAGGCGAAGCGCGCTGTTGAGGCGGGCTACTGGCACCTGTGGCGTTATCACCCGCAGCGCGAAGCGGAAGGCAAGACGCCGTTTATGTTAGATAGCGAAGAACCGGAAGAGTCGTTCCGTGACTTTCTGTTGGGTGAGGTGCGCTACGCATCCCTGCACAAGACCACCCCGCACCTCGCCGATGCCCTTTTCAGCCGTACCGAAGAAGATGCGCGTGCGCGCTTTGCGCAATACCGTCGCCTGGCTGGCGAAGAGTAATAATACTCTAACCCCATCGGCCGTCTTAGGGGTTTTTTGTCCGTGGttagttagttagcccttagtgactcTAATACGACTCACTAGAGAGAGACGCGACTTCCAGAGAAGAAGACTACTGACTTGAGCGTTCCCTCTCTGTAATACATCAAATCAATCATAGGAGGGCTAAAATGACCTCTTGTTCGTCGTTTTCTGGCGGTAAAGCGTGCCGTCCGGCCGATGACTCCGCGCTGACTCCGCTGGTGGCCGACAAGGCAGCTGCGCACCCGTGCTATAGCCGCCACGGCCATCACCGCTTCGCGCGTATGCACCTGCCAGTCGCTCCGGCCTGCAACTTACAATGCAACTACTGCAACCGCAAGTTCGATTGCAGCAATGAAAGCCGTCCGGGCGTGTCCTCTACCCTGCTGACGCCGGAACAGGCTGTGGTGAAGGTGCGCCAGGTCGCCCAAGCTATCCCGCAGCTGtcgGTGGTCGGTATTGCTGGTCCGGGCGATCCGCTTGCGAATATCGCCCGCACCTTCCGTACCTTGGAGCTTATTCGCGAACAGTTGCCGGACCTGAAACTGTGCCTGAGCACCAACGGCTTGGTGCTGCCAGATGCCGTTGATCGTCTGCTCGATGTGGGCGTGGATCACGTTACCGTCACCATTAACACCCTGGACGCAGAAATCGCAGCGCAAATCTACGCGTGGTTGTGGCTGGATGGCGAACGCTACTCCGGTCGCGAAGCCGGCGAAATTCTCATTGCCCGCCAGCTGGAAGGCGTACGTCGCCTGACCGCGAAAGGTGTGCTCGTCAAGATCAACAGCGTATTGATTCCGGGCATCAATGACAGCGGCATGGCGGGTGTTAGCCGTGCGCTGCGCGCGTCTGGTGCGTTCATCCACAACATCATGCCACTGATTGCGCGTCCGGAGCATGGCACTGTTTTCGGTCTGAACGGCCAGCCGGAACCGGACGCGGAAACCCTGGCGGCGACGCGCTCCCGCTGCGGCGAGGTTATGCCACAAATGACCCACTGCCACCAGTGCCGTGCCGACGCGATTGGCATGCTTGGTGAGGATCGCTCGCAACAGTTTACGCAATTACCGGCTCCGGAGTCCCTCCCGGCCTGGCTGCCGATCCTGCATCAGCGTGCTCAGTTGCATGCGAGCATCGCCACGCGCGGTGAGAGCGAAGCCGATGACGCCTGCCTGGTGGCCGTTGCGTCGAGCCGTGGCGATGTAATTGACTGCCATTTCGGCCATGCCGACCGTTTCTATATCTATAGCCTGTCTGCGGCTGGTATGGTTCTGGTTAACGAACGTTTCACCCCGAAATACTGCCAGGGTCGCGATGACTGCGAGCCGCAGGACAATGCCGCACGCTTTGCTGCCATCCTTGAGTTGCTGGCGGACGTCAAAGCGGTGTTTTGTGTGCGTATCGGCCATACCCCGTGGCAACAGCTGGAGCAGGAAGGCATCGAACCGTGCGTGGATGGCGCCTGGCGTCCGGTATCCGAGGTCCTGCCGGCATGGTGGCAGCAGCGCCGTGGTAGCTGGCCGGCTGCATTGCCGCACAAAGGCGTTGCGTAAACTACGAGATTTGAGGTAAACCAAATAAGCACGTAGTGGCATTAAAGAGGAGAAATTAAGCATGCCGCCATTGGACTGGTTGCGTCGTTTGTGGTTACTCTATCACGCCGGCAAAGGCAGCTTTCCGCTTCGTATGGGCTTGTCGCCGCGTGACTGGCAAGCTCTGCGCCGTCGCCTGGGCGAGGTGGAAACGCCGCTGGATGGCGAAACCCTGACCCGTCGCCGTCTGATGGCGGAGCTGAATGCGACCCGCGAAGAAGAACGCCAGCAGCTGGGTGCCTGGCTGGCCGGTTGGATGCAACAGGATGCCGGTCCGATGGCGCAGATTATCGCAGAGGTGAGCCTGGCGTTCAACCATCTCTGGCAGGACCTTGGCCTCGCGAGCCGCGCTGAACTGCGTCTGCTGATGTCTGACTGCTTCCCGCAGCTGGTTGTTATGAACGAGCACAACATGCGCTGGAAGAAATTCTTTTACCGCCAGCGTTGCCTGCTGCAACAGGGCGAAGTCATCTGTCGCAGCCCGTCTTGCGATGAATGCTGGGAACGTTCTGCGTGCTTTGAGTAATACATATCGGGGGGGTAGGGGTTTTTTGTGTCTGTAGCACGTGCATCTAATACGACTCACTAATGGGAGAGACAAGAGTCTCAATTATAAGGAGGCTTTACTACATGGCGAACATCGGCATCTTCTTTGGTACGGATACCGGCAAAACCCGCAAGATTGCGAAGATGATTCACAAACAGCTGGGCGAGCTGGCCGATGCCCCGGTTAACATCAATCGTACCACTTTGGATGACTTTATGGCTTACCCAGTCCTGTTGCTCGGCACGCCGACGCTTGGTGATGGTCAACTGCCGGGCTTAGAGGCGGGCTGCGAGAGCGAAAGCTGGTCTGAGTTTATCTCCGGTCTGGATGACGCTTCCCTGAAGGGCAAAACCGTGGCGCTGTTTGGCCTGGGCGACCAGCGTGGTTACCCGGACAACTTCGTGTCGGGTATGCGTCCGCTGTTCGACGCGCTGAGCGCCCGTGGCGCCCAGATGATTGGTAGCTGGCCGAACGAAGGTTATGAGTTTAGCGCATCGTCCGCGCTGGAAGGCGACCGCTTCGTCGGCTTGGTGCTGGATCAAGACAATCAGTTCGACCAGACCGAAGCGCGCCTGGCGTCTTGGCTTGAAGAGATCAAACGCACCGTTCTGTAATAATACATATCGGGGGGGTAGGGGTTTTTTGTGGTCATTACAACGGTTATggtctcaggagtaatacgactcactagagagagaggtcgcggacccggccgatccgggggcctcaaagccgcctcaccagatactgacaaataaaccagcgaaggaggttcctaatgtggaactacagcgagaaagtcaaggaccatttcttcaatccgcgcaacgcgcgtgttgtggataacgcaaatgcggtgggcgacgtcggcagcttatcttgtggcgatgctctccgcttgatgctgcgcgtggacccgcagagcgaaatcatcgaagaagcgggctttcagaccttcggctgcggcagcgcgattgcgtcgtccagcgcactgacggagctgatcatcggtcacaccctggcggaagcgggtcagatcaccaaccagcagatcgccgactatctggacggcttaccgccggaaaagatgcactgctctgtaatgggccaggaagctcttcgtgcggccattgctaactttcgcggtgaatcgctggaagaggagcatgacgagggtaagctgatctgcaagtgcttcggcgtcgatgaaggccatattcgccgtgctgtccagaacaacggtcttacgacgctggccgaggtgatcaattacaccaaggcaggtggcggttgtaccagctgccatgagaaaatcgagctggccctggccgagattctcgcccaacagccgcaaaccaccccggcagttgcgtccggtaaagatccgcactggcagagcgtcgtggataccatcgctgaactgcgtccacatatccaagcggacggtggtgacatggcgctgttgtccgtgacgaaccaccaagtgactgtttcgctgtcgggcagctgttctggctgcatgatgaccgacatgaccctggcgtggctgcaacagaaattgatggagcgtaccggctgctatatggaagttgttgccgcctaagaccgcgcgccccgtcagagcaatgcgtataccagctctcctgtcagcagaatggctccagtacatctaacggggcagtatccgcggcaagtcctagtccaatcgatacccgtagaccattctgaaatcgaaggaggttttccatgaaacaagtgtacctggacaacaacgcgaccacccgcctggacccgatggttctggaagcgatgatgccgtttctcacggatttctatggcaatccgtccagcatccatgacttcggcatcccggcacaagcggcgctggaacgtgcgcaccagcaagctgcggcactgctgggcgcagagtacccgtctgaaatcattttcacgagctgtgcgaccgaggccactgcaaccgccattgcgtcggccatcgcgttattgccggaacgccgcgaaatcatcacctcggtagtggagcacccggctacgctggcggcgtgcgagcacctggaacgccaaggctatcgcatccatcgcattgcggtggatagcgaaggtgcgctggacatggcccagttccgtgcagcgctctcgccgcgtgtcgcgttggtgagcgtgatgtgggccaacaacgaaaccggcgtgctgttcccgattggcgaaatggccgagcttgcccacgagcagggcgctctgttccactgcgatgccgttcaggtcgttggcaaaatcccaattgctgttggccagacgcgcatcgacatgctgtcttgctccgcgcacaagtttcatggtccgaagggtgttggttgcttgtacttacgtcgtggcacgcgctttcgtccgctgcttcgcggtggccatcaagaatatggtcgccgtgccggcactgagaatatctgtggcatcgtcggcatgggcgctgcgtgcgaactggcgaacatccatctgccgggtatgacccatattggccagttacgcaatcgcctggagcaccgtctgctcgccagcgtgccgtccgtgatggttatgggcggtggtcagccgcgtgtaccgggtactgtcaacctggcgttcgagtttatcgaaggtgaagcgatcctgctcttgctgaaccaggctggcattgccgcaagctccggctccgcgtgtacctctggcagcttggagccgagccatgtgatgcgcgccatgaacattccatacaccgcggctcacggcaccattcgttttagcctgagccgttatacgcgcgagaaagagatcgactacgtcgttgcgaccctcccgccaatcattgatcgtctgcgtgccttgtccccgtattggcagaatggtaagccgcgtccggcagatgcagtctttaccccggtttacggttaagcgactaggagcctaactcgccacaaggaaacatatggagcgcgtcttgatcaacgatactaccctgcgtgatggcgaacaatctccgggcgtagcgtttcgtacctccgagaaagttgccatcgcggaggcactgtacgctgcgggtatcaccgcgatggaagtcggcactccggcgatgggtgatgaagagatcgcccgcattcagctggtgcgtcgtcaactgccggacgcgacgcttatgacctggtgccgtatgaacgctctggaaatccgtcagagcgcggatctgggtattgactgggtggatatctcgatcccagcatccgacaagctgcgtcagtacaagctgcgtgagccgctggccgtgctgctggagcgccttgcgatgtttatccatctggcccacacgttaggcctcaaagtatgtattggttgcgaggatgcgagccgtgcgtctggtcagaccctgcgcgccattgccgaggtggcccagcaatgcgcggctgcgcgcttgcgttacgctgacaccgtgggcctgctggacccgttcaccaccgcagcccagatcagcgccctgcgtgacgtttggtcgggcgagatcgagatgcatgctcacaatgatctgggcatggctaccgcgaacacgctggcggcagtttcggctggcgccacgtcggtgaacactaccgtcctcggtctgggtgaacgtgcaggcaacgcagccctggaaaccgttgcgctgggcctggaacgctgcctgggcgtggaaaccggcgtccatttcagcgcgctcccagcgctctgtcagcgcgtcgcggaggctgcacagcgcgcaatcgacccgcaacagccgctggtgggtgaattggttttcacccacgaatctggtgttcacgttgcggcgctgctgcgcgacagcgaatcctatcaatctattgccccaagcctcatgggccgtagctaccgtctggtgctcggcaagcattcgggtcgtcaggctgtcaacggtgttttcgaccagatgggttaccacctgaatgcggcgcagatcaatcagttgctgccggccattcgccgcttcgccgagaattggaaacgctctccgaaagactacgaactggttgcgatctatgacgaattgtgcggtgaatccgcccttcgtgctcgcggctaaccgatagtttcaagagaaagggagtagaaacagaatggagtggttttaccagattccgggtgtagacgaattgcgcagcgctgaatccttctttcagttcttcgcggttccataccagccggaactgctgggccgctgctcgcttccggtgttagcgacgttccaccgtaaactgcgtgcggaggtcccgctgcaaaaccgtctggaggacaatgatcgtgcgccgtggctcttggcgcgccgcctcctggccgaatcttatcagcagcaatttcaggagagcggcacctaattcaccagcccgaatcaatataggtcatacaatgcgcccgaaattcaccttctctgaagaggtccgcgtagttcgcgcgattcgtaatgatggcaccgtggcgggttttgcgccaggtgcgctgctggttcgtcgcggttcgacgggctttgtgcgtgactggggtgtgttcctgcaagaccagatcatctatcaaatccactttccggaaaccgaccgcattatcggctgtcgcgagcaggagttaatcccgattacccagccgtggttggctggtaacctccagtatcgtgacagcgtcacgtgccaaatggcactggctgtcaacggtgacgtggttgtgagcgccggtcaacgtggccgtgtggaggccactgatcgtggcgaacttggcgattcctacaccgtggacttcagcggccgttggttccgcgttccggtccaggccatcgcgctgattgaagagcgcgaagaataatcagagactgaagttattacccaggaggtctataatgaatccgtggcagcgctttgcccgtcaacgccttgctcgcagccgctggaaccgtgatccggctgctctcgacccagccgataccccagcgttcgagcaggcgtggcagcgtcaatgccatatggaacaaaccatcgtagcgcgtgtcccggaaggcgatattccggctgccttactggaaaacatcgcggccagcctggcgatctggctggacgagggtgacttcgctccgccggagcgcgctgcgattgtgcgtcatcatgcacgtctggagctggcgtttgccgacattgcccgccaggcaccgcaaccggatctgagcacggttcaagcgtggtatctgcgtcaccagacgcaattcatgcgtccggagcagcgtctgacccgtcacctgctcctgacggtcgataatgatcgcgaggcggtgcatcaacgcatccttggcctgtatcgtcagatcaacgcgagccgtgacgccttcgccccactggcacagcgccactctcattgcccgtccgccttggaagaaggccgtctgggctggatctcccgtggtctgctgtacccgcagctcgaaaccgcgttgtttagcctggcggaaaacgcactgtcgctgccgattgcgtcggaattgggttggcacctgttatggtgcgaggccattcgtccggcagccccgatggagccgcaacaggcccttgaatctgcgcgcgactacttgtggcagcagagccagcagcgccaccagcgtcaatggctggagcagatgatttcccgccaaccgggcctgtgtggttaaTAGCATAACCCCttggggcctctaaacgggtcttgaggggttttttgtPaenibacillus WLY78 nif cluster (SEQ ID NO. 3)gtagggcgcattaatgcagctggactagtGAATTGAGGATAAATGTCAGGGATTTCATGGAGAAGTGAATTGACTGTATTTGTCCCTGTCTCTAAGATGTAATTATATTCCAGACAAAAACAGAGATTTATGTAAGGGAATATAACGTAGAGAGGAGGGAATGAATGGACTCTTTAGCTGATCTCTCGGAAACCCCCTTAGCATTAGAAACTCTCAGACGACATCCCTGTTATAACGAAGAGGCACATCGCTATTTTGCGCGCATTCATCTTCCAGTAGCCCCGGCATGCAATATTCAGTGCCATTATTGCAACCGCAAATTCGATTGCGTCAATGAAAGCCGTCCCGGCGTTGTTAGTGAACTGCTTACGCCGGAGCAGGCGGCGAGCAAGACCTATGGCGTAGCGGCACAGCTGATGCAGCTGTCCGTTGTCGGCATTGCGGGACCTGGAGATCCGCTGGCCAATGCGGAGGCAACCTTCGATACCTTCCGCCGGGTCCGTGAGACAGTTAAGGACGTCATATTCTGTCTCAGCACGAATGGCCTTACTTTGATCAGGCATATCGACAGGATTGTAGAGTTGGGTATTTCGCATGTCACGATCACGATCAATGCTGTAGATCCAGTGGTGGGGAGCCGCATTTATGGATGGGTCTACGATGAAGGAAAACGCTATGCGGGTGAGGAGGCCGCACGACTGTTGATTGACCGCCAGCTGGCAGGCTTGAAGATGCTGGCTTCGAGAGGTGTATTGTGCAAGGTGAACTCGGTGCTGATTCCCGAAGTCAATGATGCCCATCTGCCGGAGGTAGCGAGGGTGGTCAAGGAGCACGGCGCGGTGCTGCACAACATTATGCCGCTCATCATCGCACCTGGTAGCCGATATGAGCAGGAAGGGATGCGGGCACCCCGTCCCCGTCTGGTCCGGCAGCTGCAGGAGCAATGTGCTGAAGCGGGAGCTGTCATTATGCGCCATTGCCGTCAGTGCAGGGCGGATGCGATTGGACTGCTGGGCGAGGATCGCAATCAGGATTTTACATGGGAGAACATTGCTGCTGCTCCTCCCATGGATGAAGAGGCAAGGGCACAATTTCAGAAAGAACTGGATGAGAAGGTGAGAGTGAGAATGGAACGCAAGGAGGGACAATCGCACCACAAACAACCGTCAACCGGTGCTGGTTGTAGCTGCCCGTTATCTGGGGATAAGCCTGAAGCGAGCTTTACCTCAAAGCCGGTCTTAATCGCTGTGGCTAGTCGTGGTGGAGGGAAGGTGAATCAGCATTTCGGTCGTGCCAAGGAATTTATGATCTATGAAAGCGACGGGACCATCGTAAATTTCATAGGCATTCGTAAGGTGCAATCCTACTGTCACGGGAAAGCCGATTGCAATGGAGATAAGGCCGAGACGATCAAGGAGATCCTTTCCATGGTACATGATTGTGCATTGCTGCTGTCGTCCGGCATAGGCGAAGCCCCCAAAGAGGCATTGCAGGAAGCGGGCGTGCTGCCTATTGTGTGCGGCGGGGATATTGAGGAGTCCGTTCTGGAATATGTAAAATTTCTGCGTTATATGTATCCTGTGCAGACGGGTAAGGGAAGTAAGCGTAATAAGGGAGTTAAGGGCAATCATTCGGATTTACCCATTGAACATTTTGGAGGCTGAGAAAATATGAGACAAATTGCGTTTTACGGTAAGGGCGGTATCGGCAAATCGACAACCTCGCAGAATACACTGGCTCAACTTGCGACCAAATTCAAACAAAAAATTATGATCGTAGGCTGTGATCCCAAGGCAGACTCCACCCGTCTTATTTTGAATACGAAGGCCCAACAGACCGTACTGCATCTGGCAGCTGAAAGGGGTACGGTGGAGGACTTGGAACTGGAGGATGTTGTCCAGAAGGGCTTCGGTGATATTCTGAACGTGGAATGCGGCGGGCCAGAGCCCGGTGTCGGCTGTGCAGGACGCGGTATCATCACAGCCATTAATTTTCTGGAGGAAGAGGGGGCCTACGAAGGGCTGGATTTCGTTTCCTACGATGTACTGGGGGACGTCGTGTGCGGGGGGTTCGCCATGCCGATCCGGGAGAAGAAGGCGCAGGAAATCTACATCGTATGCTCAGGCGAGATGATGGCTATGTACGCTGCCAACAATATTGCGCGCGGGATCTTGAAGTATGCCAACAGCGGCGGGGTGCGTTTGGGCGGCTTAATCTGCAACAGCCGGAATACGGACCTGGAAGCGGAATTGATCACAGAGCTTGCAAGAAGATTGAACACGCAGATGATCCACTTTTTGCCGCGTGACAATGTTGTGCAGCACGCTGAGCTGCGCCGTATGACCGTTACCCAATATAACCCGGAACATAAGCAGGCTGCGGAGTATGAAGAGCTGGCAGGTAAGATTTTGAATAATGACATGCTAACGGTTCCCACGCCCATTTCCATGGAAGATCTGGAGGATCTATTGATGGAATTCGGCATTATTGAGGATGAAGAAACCGCAATTAACAAAGCTGAGGCGTCCGGGCAGTAGGCTGTAGCCAGAAGGCTTAATGACGGAACCATCGTGTAATGATGGGAGGAGCTGAACGCGCAGCTCGCAGGAGGGAGGAATAGGCCAAATGAGCAGTATTGTGGATAAGGGTAAGCAGATCGTAGAGGAGATACTGGAGGTATATCCCAAGAAGGCCAAGAAGGATCGGACCAAGCATTTTGAGATCGCGGATGAGGAGCTTGTGAACTGCGGAACCTGTTCCATCAAGTCCAACATGAAATCACGGCCTGGCGTCATGACAGCAAGGGGCTGTGCTTATGCAGGCTCCAAGGGTGTGGTATGGGGCCCGATTAAAGACATGGTGCACATTAGCCATGGTCCCATCGGCTGCGGACAGTACAGTTGGGGTACCCGACGCAATTATGCGAATGGGATATTGGGAATCGATAATTTTACCGCCATGCAGATTACAAGCAATTTTCAGGAAAAAGATATCGTGTTCGGTGGAGATAAGAAGTTGGAGGTGATCTGCAGGGAAATTAAGGAGATGTTCCCGCTGGCTAAGGGTATCTCCGTGCAATCTGAATGTCCGGTCGGACTGATTGGTGATGATATCGGGGCCGTGGCCAAGAAGATGACAGAGGAGCTGGGCATTCCGGTCATTCCTGTACGCTGTGAGGGCTTTCGCGGGGTGAGTCAGTCTCTGGGCCATCACATTGCCAATGATGCTATCCGCGATTTTCTAATGGGGCGCCGAGAACTGAAGGAGTGCGGGCCTTATGATGTCTCCATTATCGGAGACTACAATATCGGCGGTGATGCCTGGGCGTCGCGCATTTTGCTGGAGGAAATGGGACTGCGGGTCATAGCGCAGTGGTCGGGTGACGGTACGATCAATGAGCTGGGGATTGCGCATAAATCCAAGCTCAACCTGATCCATTGTCATCGTTCCATGAATTATATGTGCACAACAATGGAGCAGGAATACGGAATTCCCTGGATGGAATATAACTTCTTCGGCCCGACCAAGACGATGGAGAGCCTCAGAGCGATTGCTGCCCGCTTCGACGAGACGATTCAGGAAAAATGTGAGCAGGTCATCGCCCAATATATGCCGCAGATGGAGGCGGTCATCCGTAAATATCGCCCACGTCTGGAAGGTAAAAAGGTGATGCTTCTGATTGGCGGGCTGCGGGCAAGGCATACCATCGGGGCCTATGAGGATCTGGGTATGGAAATTGTGGCTACAGGCTATGAATTTGCCCATAAGGATGATTACGAAAAGACGTTTCCCGATGTAAAAGAAGGCACCATTCTGTACGATGATCCAACGGCATATGAGCTGGAGGAACTGGCCCAGCGGCTGAATATTGACTTAATGGGCGCCGGAGTCAAGGAGAAATACGTGTATCACAAAATGGGCATTCCCTTCCGTCAAATGCACTCCTGGGATTACAGCGGGCCTTATCATGGTTTTGACGGCTTTAAGATTTTTGCACGTGATATGGATATGACCATAAACAGTCCAGTATGGAGCCTGCTGCCCTCACGGCAGACTGCGGAGGTGCCGGTATGAGCGAGCGTCCGAATATTGTCGATCACAATCAGCTGTTTCGGCAGGATAAATATGTGCGCCAGCGTGAAGAAAAACGAGCCTTCGAGGCCCCATGTTCGCCGGAGGAGGTTACCGACACCCTGGAGTACACCAAGACCAAGGAATACAAAGACAAGAATTTTGCCCGTACAGCCGTAGTCGTGAATCCGGCCAAGGCTTGTCAGCCGCTGGGAGCGGTTATGGCTGCACTGGGCTTCGAAAAAACGCTCCCGTTCATTCATGGTTCACAGGGCTGTACGGCTTATTTTCGCAGTCATCTTGCCCGCCACTTCAAAGAGCCTGTTCCTGCCGTCTCCACCTCGATGACCGAGGATGCCGCCGTATTCGGCGGCATGCGCAACCTCATTGACGGTATAGAGAACTGCATTGCCTTGTATCAGCCGGAGATGATTGCGGTATGCACGACCTGTATGGCAGAGGTGATCGGGGATGATCTGTCTGCCTTCCTGGCCAATGCCCGTCAGGAGGGAGTCCTTCCTGAGGATATGCCAGTTCCTTTTGCCAATACCCCCAGCTTCTCTGGTTCACATATTACAGGCTATGACGCCATGCTGCGCTCTGTACTGGAGACGCTGTATAACAAGTCAGGCCGGACGGCGCAGCCTGGTCATGAATTGAAGCTGAATGTACTGCTCGGGTTTGACGGGTATACGGGCAATTTTGCGGAAATGCGGCGCATGCTGGGGATGTTCGGCGCTACGTATACCATTCTGGGTGACCACAGCAGTAATTTTGATTCAGGGGCCACTGGAGAGTACAGCTACTATTACGGGGGAACGCCGCTTGAGGATGTGCCTAAGGCCGCAGATGCTGCCGGCACGTTGGCGATTCAGCAGTACTCTCTTCGTAAAACACTAGGCTATATGAAGCAAACCTGGGGGCAGCAGGTGTCCTCCATCTCCACACCGCTGGGCATCCGGGCTACAGATCGCTTGCTTGAGGAGATTAGCCGCCTGTCTGGAAGGGAAATTCCCGAGGCATTGAAGCAGGAGCGCGCCCGAATTGTGGATGCCATGATGGATTCACATGCTTATCTGCACGGCAAACGAGTGGCTATGGCAGGAGACCCGGACATGCTCATCGGCTTGATTGGCTTTTGTCTGGAGCTGGGCATGGAGCCGGTGCATATTGTTTGCTCCAATGGGGACCGAAAATTTGAGAAGGAAGCAGAGCTTCTGCTGAAGTCCAGCCCTTACGGTGCAGAAGCCACGGTTCATTCCGGTCAGGATTTGTGGCATATGCGTTCGCTGCTGTTCCAGGACCCGGTGGACCTGGCTATTGGCAGCTCCCATCTGAAGTTTGCAGCGAAAGAGGCGGAAATTCCTTTGCTTCGTGTAGGCTTTCCGATCTTCGACAGGCATCATCTGCATCGTTATCCGATTATCGGCTACCAGGGTGCGCTGAATCTGCTCACCCAATTCGTGAATACCATACTAGATGTCATGGAGGAGCAGGCTCCGGATCATAGCTTTGATCTGGTGCGCTAATTGCTGTATCGCGTAGAAGGAAGTTGACAGCTTGGCTTGTGATTTCAATGGATTCTATCTGAAATAAGGGGGTGTGTGGATGGAGCCGGCTGTGTCTAACGGAAGGCTGGAGGTATCCTGCGGCAATAAAATTCCCAAAAGCACGCCCTGTCCCCGGCCTGTGCCGGGAGAGGCTTCGGGTGGCTGCTCCTTTGACGGGGCCCAGATTACACTGATCCCCATTGCAGATGCGGCTCATCTGGTGCACGGGCCAATTGCGTGTCTCGGCAATAGCTGGGAGAGCAGAGGCAGTCTGTCCAGCGGCCCAGAGCTGTCGGCTTATGGCTTCACTACTGATCTTGGAGAACAGGACATCATTTTTGGTAGTGAACAGAAGCTGCATGAATCGATCCGCTACATTGTCAGCCGCTTTGCTCCTCCCGCTGTGTTTGTCTATACCACATGTGTCACAGCCCTCACTGGTGAAGATATCGAGGGGGTTTGCAAGGCTGAATCGGAGCGGCTGGGGACGCCGATCATTCCGGTGAACAGTCCGGGATTTGTGGGCAGTAAGAATCTCGGAACCCGGCTGGCCGGAGATGTGCTGTTCCAGCATATTATCGGCAGCACCGAGCCGGAACAGACAACCTCCCATGATATCAATCTCATTGGGGAATACAATATTGCGGGCGAGATGTGGCATATCGAGCGGCTGATGCAGCAGGCGGGAATGAGTATCCTGTCCCGAATTACCGGGGACGGTCGGTTCCGCGAGGTGGGCTGGGCGCACCGTGCCAAGGCCAACATGGTCGTATGCAGCCGGGCTTTGCTGGGTCTGGCAGTCCAAATGGAGCGTAAATACGGCATTCCTTATTTTGAAGGTTCATTTTATGGAGCAAAGGAGACGAGTTATTCCTTGCGGCAGATGGCTTACCTGACCGGAGATCGTGATGTGGAGCGACGGGTGGATAAGCTGGCCGCACGGGAGGAAATGAGGCTATCGCTGGAGCTGGAGCCCTACCGCAAGCAGCTGAAAGGAAAGCGGGCAGTGCTCTATACCGGGGGTGTAAAGAGCTGGTCTGTCATTACGGCTTTGCAGGAGCTGGGCATAAAGGTGGTTGGTGTAGGCACGAACAAGAGCACTGCCGAGGATGTATCCCGGATTGCTGACCGTATCGGGGATGATGCAGAATACATCCCGGAAGGAGGCGCCCGGCAGATTCTCAAGACCGTACGGAGCCGCAAGGCAGACATGGTCATTGCCGGAGGCCGGAACATGTATATGGCGCTTAAGGAACAGATTCCTTTTGTGGACATCAATCAAGAGCGGCACAAAGCCTATGCGGGCTATGACGGGCTGTTGTCTCTGGCGAAACAGCTTGTGCATACGCTGCAGCATCCAGTATGGGAGCTGACCGCCAAATTGGCTCCATGGGAGGAGGAGACGGAATTTGCTGATTAAATCCGCCACGAAGCCTGTCAGTGTCAACCCGCTCAAGGTAGGACAGCCTTTGGGCGGCGTGCTGGCTCTGCAGGGGATGTATCGCTCAATGCCTTTGCTGCACGGCGCTCAGGGCTGCTCGGCCTTCTCCAAGGCGCTGCTGACTCGCCATTTTCGAGAGCCGATTGCCGTTCAGACCTCTGCGTTGCAAGAGATGGACGTTATATTTGATGCAGACCGGAATCTGGAGGAGGCGCTGGATCATATCTGGTCCAAACACCATCCAGATGTCATCGGCGTTATCAGCACGGCCCTCACTGAGGTGGCAGGCGTTGACTTTCAGTCTAGGGTAAAGGCGTTCAAGCGAGAACGGGCATTGAAGGACAGTCTGCTGTTTTCTGTATCGCTGCCTGATTTTCACGGCTCTCTGGAGACGGGCTACAGCAGTACAGTAGAGTCACTAATGGATGCCGTACTCGGGTTGGCCGGGGGCAAGTCCCCCAAAAAACAGCGCCGGACGCAGGTCAATCTGCTGCCGGCTTCTTATCTGACTGCCGGAGATGTCATGGAAATCAAGGATATTATCGCTTCCTTCGGCCTGGAGGTTATTACGCTCCCCGATATTTCCACTTCCTTGTCCGGTCACCTGCTGACAGGCTTTTCCCCTTTGACGAGAGGGGGGACTCCGCTGGATTCAGCCTGCCAGATGCTGGAGTCTTCCTGCACCATTGCCATTGGCGCGAGCATGGAGCGTCCGGCGCGCAGGCTGACTCATGCTGCAGGTATTCCCTACCACTTGTTCGCTGGTCTGTCTGGCTTGGCCGCGAGTGATGCGTTCATACATTTTCTGCAGAAAATCAGCCGCGAGCCAGCCCCCGTTCGCTTCCGTTGGCAGCGTGAAAATCTGTTGGACAGCATGCTGGATGCCCATTTCTATTATTCTGGCGCTTCGGCTGTAGTGGCGCTAGAACCGGATCATATGCTGTCGACCGCAGCCTGGCTGGAGGAGATGGGAGTGGAACTGAAGCGGCTAATTACACCCTGCAGCACGCCCGCACTGCAAAAGACAGAACGGGAAGTCTGGATCGGTGACCTGGATGATGCAGAGGAGAGCGCGCAGGGTGTTGATTTGTGGATCAGCAACTCACATGGAAGAAAGGGAGCGGCACGGGCTGGGGCCTCATTCGTACCGGCAGGCTTGCCGGTGTATGACGAGCTAGGCGCCCACACATCCGTAAGCGTCGGATACCGTGGAACCATGGAGTGGGTGAACAAAGTAGGCAATGTATTGCTTGCCGAGAGGGGGAGGGGAGGATGAAGGTTGCATTTGCGACGGAAGACGGCGTGCTTGTGAATGCTCATTTTGGGCAGAGTCCCATGTTCACTATATTCGAAATCCGGCACTCAGGCGTCCAGTTCCTGGAGCATCGGCGGATAGCCCTGGGGAGCGATGAGAATGAGGCGGGCAAGATCGCCAGCCGAATTGGCCTGATCGAGGATTGTGCCTTGATCTTCCTGGTACAGATTGGCGCTTCCGCCGCCGCACAGGTTACCAAGCGGACCATTATGCCTGTGAAGGTGGCCTTCGGTAGCACCATTGAGGAGCAGGTCCAGCGTCTCCAGAATATGCTGACTCGCAATCCGCCCATGTGGCTTGCCAAAATCCTGCATGCTGAGGAGGGCAGCGGCAAAGCCGAATCATGAGCCCTCCTGTAAGGAAGAGCAACCATATAGGGTATTAAGATCCTGCAGACCGAATATCTTAAAGGCGGGAGCCGCACATGGAGGGGGTGGACGAATGGTACAACTGCTGGAAGACAGTAGATACGGACGCCAGTTGAAGCTGCTGGGAGTGGAAGGTCAGAACAGGCTAAAGCAGGCTACGGTTATGGTTGCAGGCATCGGAGGATTGGGAGGGGCAGCGGCCATGTACCTGGCCGCTGCCGGAGTAGGAAAGCTGATATTGGCCCATGAGGGCGTAATCCATCTGCCCGATATGAACCGGCAGGTGCTGATGGACAGCGGACGAATCGGGGAGGAACGGATGGAGACGGCATTACAGCATTTGCATCGTATCAATCCGGAGACCGAGCTTGAGGGCCACGCCCACAGAATCACGGAAGAATCCTCTGGACCATGGGTAGAAGCGTCGGATATCGTGATTGATGCACGATATGACTTTCCGGAAAGATATGCGCTGAACAGACTATGTGTTCGACATGGAAGACCGATGATAGAAGCGGCCATGTACGCCTATGAAGTATCATTGATGACCATTGATCCCGGTAAGACGGCATGCCTGGAATGTCTTTACCCGGAAGGCGGACAGCCTTGGGAACCTCTGGGATTCCCGGTCCTGGGAGCCACCTCCGGCTTGATTGGCTGCATGGCTGCACTGGAAGCGGTCAAATGGATTACAGATGCGGGCAATCTGTTCACTGACCGCATGTACCGTATGAATGTGCTGGATATGAGCAGCTGCACCATAGCGGTCAAACGCAACCCGCGTTGTCCGTGCTGCGGAACGGGAGGGGATACAGATGAGTCGGTTGCATATTTGTGATACGACACTTCGTGACGGAGAACAGGCTCCGGGCGTTGCCTTTTCAGCCGAGGAAAAAACTGAAATTGCCATCATGCTGGACTCGGCGGGGGTGGAGCAGGCTGAGATCGGAATTCCGGCAATGGGAAAGACGGAGTGCAGGTCTATTGCCAGGATTGCTGCTCTCGGACTTCAGATGAAGCTAATGACCTGGAATCGCGCGGTGTTCACGGATATTGATGCAACTGAATCGACAGGTGTCGGCTGGGCCCATATTTCGGTTCCCGTGTCGACGGTGCAGATGAAGTCCAAGCTGGGTATGAATCCTGAGCAGGTGACGGAGCTGATCCGCAAGTCTGTCGATTACGCTCTGTGTAAAGGATTGACTGTTTCCGTAGGCTTTGAGGATGCTTCAAGGGCAGATGACCTGTTCCTTGAGCAGTTGGCGAATCAGCTCTATAGGGATGGCATCCGGCGCTTCAGATATGCCGATACGCTGTCCGTTCACCATCCCGCTGCCATAGCTGCCCGTATAGACAGGCTTGTATCGCGCGTGCCACAGGATGTGGAGCTTGAGATTCACTGTCATAATGATTATGGCCTGGCGCTTGCCAATACCCTGGCAGCTTTGCAAGCGGGAGCTGTCTGGGCCAGTACCACGGTGTCGGGACTTGGGGAAAGGGCAGGTAATACCGCGCTGGAGGAGGTGGTGATGTCGTGGAGGGACCTATATCAAGGAACCTGCAGCGTCCGTCCCGAACTGCTGAACCCGCTGGCTGCACTGGTGTCCAAAGCCTCCAACCGAATCATTCCTGAAGGCAAGCCCATTGTGGGAGACATGGTATTCGCCCATGAATCCGGCATACATATCAACGGTCTGCTAAAGGAGCGCGCCGCCTATCAGGCGCTTGATCCGACTGAGCTGGGCACTGACCATTCCTTCGTACTCGGCAAGCATTCGGGCAGAAGTGCAGTTCAATATATGCTGGAGCAGGAAGGAATCGAGGCAGGCTCCGGTGAAATCAAGTTCCTGCTGGAGCGGCTTCGCCTAGTCGGTGAAGATCCCAAGCGTGTCATCCATAGCGCGGATTTAAGACGCTGGCTGCAGTATTATCCGGCAGAGCTGCCGAAATAACCGAAAAAGCGTTCCCGTCCGGTAAGTGTGACCGTGACTGGAACGCTTT Klebsiella oxytoca M5a1 nif cluster (SEQ ID NO. 4)GAATTCTAGACTGCTGGATACGCTGCTTAAGGTCATGCAGCAGGAGAACTAAAGGCCCGCTACTCCTCGCCGGCCAGCCGCCGATACTGGGCAAAGCGGGCCCGCGCGTCCTCCTCGGTTCGGCTAAAGAGCGCATCCGCCAGATGCGGCGTCGTTTTGTGCAGCGAGGCGTAGCGCACTTCGCCAAGCAAAAAGTCGCGGAAGCTCTCCTCCGGCTCTTCGGAATCGAGCATAAACGGCGTCTTACCTTCCGCTTCCCGCTGCGGATGATAGCGCCACAGGTGCCAGTATCCCGCCTCAACCGCCCGTTTCGCCTCGCGCTGGCTGCAGCGCATACCGGCTTTCAGCCCGTGGTTAATGCAGGCGGCGTAGGCAATCACCAGCGACGGTCCCGGCCAGGCTTCGGCCTCGGCGATCGCCCGTAGGGTCTGATCTTTATCAGCGCCCATCGCGACCTGGGCCACGTACACATTGCCGTAGCTCATCGCCATCATGCCGAGATCTTTTTTCCGCGTGCGTTTGCCCTGCGCGGCAAACTTCGCGATGGCCGCCACCGGGGTCGATTTAGACGACTGGCCGCCGGTATTGGAGTAAACCTCGGTGTCAAACACCAGAATATTGACGTCTTCCCCGCTCGCCAGCACGTGATCGAGACCGCCGAAGCCGATATCGTAGGCCCAGCCGTCGCCGCCGAAAATCCACTGCGAACGACGAACAAAATAGTCGCGGTTCTGCCACAGCTGCTCCAACAGCGGCACGCCCTCTTTTTCCGCCGCCAGCCGTTCGCTGAGCCGGTCCGCGCGCTCGCGGGTGCCCTCGCCTTCATCCTGCTTCGCCAGCCACTGGCGCATTGCGTCGCTAAGTTCGTCGCTGACCGGTAGCGCCAGCGCGGCGGTCATATCATCGGCGATTTGTTGACGCACCGCCTGGCCGCCGAGCATCATGCCGAGGCCAAACTCCGCATTATCCTCAAACAGCGAGTTCGCCCATGCCGGGCCATGGCCGCGGTGGTTGGTGGTATAGGGAATCGACGGCGCGCTGGCTCCCCAGATAGAAGAGCAGCCGGTGGCGTTAGCGATCAGCATCCGGTCGCCAAACAGCTGGGTTATCAGGCGGGCATAAGGCGTTTCACCGCATCCCGCGCAGGCGCCGGAAAACTCCAGCAGCGGGGTTTCAAACTGGCTGCCTTTGACCGTCGTCTTACGAAACGGATTGCTCTTCGGCGTCAGCGCCAGCGCATAGTCCCAGACCGGCGCCATCTGACGCTGGCTATCGAGAGACTGCATTTTTAACGCCTTGCCGCGCGCGGGACAGATATCCACGCAGTTGCCGCAGCCGGAACAATCCAGCGGCGAGATAGCCAGATGGTAGTGATACTCCTTCGCTCCCTGCGCGGGTTTGCTCAGCAGCCCAACCGGCGCGGCGTCATGCTCTTCGCCGTTGAGCAGCGCCGGGCGGATCGCCGCATGCGGGCAGATAAAGGCGCACTGGTTACACTGCGTGCAGCCCTCCGGCTGCCAGACCGGCACTTCCAGCGCGATCCCGCGTTTCTCCCACGCGGCGGTGCCCGAAGGAAAGGTCCCGTCCTCCATACCGACGAACGCGCTCACCGGCAGCTGGTCGCCGCACTGGCGGTTCATCGGCTGCAGAATATCGCGGATGAAATCCGGCATCATGGCTGATGCTTGCGCCGCGGGTTCATCCAGCGTCGCCCAGTGCGCCGGAATCGTCACCTGATGCAGCGAGGCCATGCCCAGCTCGATCGCCCGCTGGTTCATCTCAATCACCGCCGCCCCTTTGCTGCCGTAGCTTTTTTCAACCGCCTGCTTGAGGTAATCCGCCGCGGTCTGCGGGTCGATAATCGCCGCCAGCTTAAAGAACGCCGCCTGCATCAGCATATTAAAGCGCCCGCCCAGCCCGAGCTCGCGGGCGATATCCACGGCGTTCAGGGTATAAAAATGGATATTTTCCCGCGCCAGATAGCGTTTAAAGCCGACCGGCAGATGCTGCTCCAGCTCCGCATCGGACCAGCTGCAGTTGAGTAAAAAGGTCCCGCCCGGCTTTAATCCGTCCAGCAGATCGTAGCGCTCAACGTAGGACTGCTGCGAACAGGAGATAAAATCGGCCCGATGGATCAGGTAGGGCGAATTGATCGGCCGGTCGCCGAAGCGTAAATGTGAAACGGTAATGCCGCCGGATTTTTTCGAGTCATAAGAAAAGTAGGCCTGCGCGTAGAGCGGCGTTTTATCGCCGATAATTTTGATCGCGCTTTTATTGGCCCCGACGGTGCCGTCCGAGCCCATGCCCCAAAATTTACAGGCGGTGATGCCGTCATGCGAGACCGCCAGCGTCTGCTGGCGCGGCGGTAACGAAGTAAAGGTTACATCATCGACAATCCCGAGGGTAAACCCGTCCATCGGCAGCGGTTTATTGAGGTTATCAAAGACGGCCGCGATATCGTTGGGCAGAACATCCTTCCCGCCAAGCGCATAGCGGCCGCCGACGATTAGCGGCGCATCGTCGTGGTGGTAGAAGGCGTTTTTCACATCCAGGCACAGCGGTTCAGCCTGAGCGCCGGGCTCTTTGGTACGGTCAAGGACGGCAATCCGCTGCACGGTTTTCGGCAGCTGGGCGAAGAAGTGGGCCAGCGAAAAAGGGCGAAACAGATGCACGCTGAGCAGCCCGACCTTCTCTCCCGCCGCGTTCAGCGTATCCACCACTTCCTGAACGGTATCGCAGACCGATCCCATTGCGATAATCACCCGTTCGGCATCCGCCGCGCCGGTATAGTTAAACAGATGATACTCCCGGCCGGTGAGCGCGCTGATTTGCGTCATATAGCTTTCGACAATGTCGGGCAGCGCCTGATAAAAACGGTTGCCCGCCTCCCGCTCCTGGAAGTAGATATCCGGGTTCTGCGCCGTTCCGCGGATGACCGGATGATCCGGATGCAGCGCGTTACGGCGGAAGCTGTCGAGCGCGGGCCGGTCCAGCAGCGTCGCCAGCTGCTCATATTCCAACACCTCGATTTTTTGAATTTCGTGCGAGGTGCGAAAACCGTCGAAGAAGTTAACAAACGGGATGCGTCCCTTAATCGCCGCCAGATGCGCCACCGCCGACAAATCCATCACCTGCTGCACGTTGTTCTCCGCCAGCATCGCGCAGCCGGTCTGGCGGACCGCCATCACATCCTGGTGATCGCCAAAAATATTCAGCGAATTGGTCGCCAGCGCCCGGGCGCTGACGTGAAAGACGCCCGGCAGCAGTTCACCGGCGATTTTGTACATGTTGGGGATCATCAGCAGCAGCCCCTGGGAGGCCGTATAGGTGGTGGTGAGCGCCCCGGCCTGCAGCGCGCCGTGGACCGCGCCTGCCGCGCCGGCCTCCGACTGCATCTCCATTAAGCGCACCGGCTGGCCAAAAAGGTTCTTTTTCCCCTGCGCCGCCCACTCGTCGACGTTTTCCGCCATCGGCGTGGAGGGGGTTATGGGGTAAATCGCCGCGACCTCGGTAAAGGCATAAGAGATCCAGGCCGCCGCGGCGTTGCCATCCATTGTTTTCATTTTTCCGGACATTGTTCAATCCTCGAAGGTGAGAGGCATCTTCGCCGCCTCAAATAAGCGGCAAACCCAGTTGTTGCCTCAAGCACAGCCTGTGCCAGCTCGCGGATGACAGAAGAGTTAGCGCGAATTCAACGCGTTATGAAGAGAGTCGCCGCGCAGCGCGCCAAGAGATTGCGTGGAATAAGACACAGGGGGCGACAAGCTGTTGAACAGGCGACAAAGCGCCACCATGGCCCCGGCAGGCGCAATTGTTCTGTTTCCCACATTTGGTCGCCTTATTGTGCCGTTTTGTTTTACGTCCTGCGCGGCGACAAATAACTAACTTCATAAAAATCATAAGAATACATAAACAGGCACGGCTGGTATGTTCCCTGCACTTCTCTGCTGGCAAACACTCAACAACAGGAGAAGTCACCATGACCATGCGTCAATGCGCTATTTACGGTAAAGGCGGTATCGGTAAATCCACCACCACGCAGAACCTCGTCGCCGCGCTGGCGGAGATGGGTAAGAAAGTGATGATCGTCGGCTGCGATCCGAAGGCGGACTCCACCCGTCTGATTCTGCACGCCAAAGCACAGAACACCATTATGGAGATGGCCGCGGAAGTCGGCTCGGTCGAGGACCTCGAACTCGAAGACGTGCTGCAAATTGGCTACGGCGATGTGCGCTGCGCGGAATCCGGCGGCCCGGAGCCAGGCGTCGGCTGCGCGGGACGCGGCGTGATCACGGCGATCAACTTTCTTGAAGAAGAAGGCGCCTACGAGGACGATCTCGATTTCGTGTTCTATGACGTGCTCGGCGACGTGGTCTGCGGCGGCTTCGCCATGCCGATCCGCGAAAACAAAGCCCAGGAGATCTACATCGTCTGCTCCGGCGAAATGATGGCGATGTACGCGGCCAACAATATCTCCAAAGGGATCGTTAAATACGCCAAATCCGGCAAGGTGCGCCTCGGCGGCCTGATCTGTAACTCACGTCAGACCGACCGTGAAGACGAACTGATTATTGCCCTGGCGGAAAAGCTCGGTACCCAGATGATCCACTTTGTGCCCCGCGACAACATCGTGCAGCGCGCGGAGATCCGCCGCATGACGGTTATCGAGTACGACCCCGCCTGTAAACAGGCCAACGAATACCGCACCCTGGCGCAGAAGATCGTCAACAACACCATGAAAGTGGTGCCGACGCCCTGCACCATGGATGAGCTGGAATCGCTGCTGATGGAGTTCGGCATCATGGAAGAGGAAGACACCAGCATCATTGGCAAAACCGCCGCCGAAGAAAACGCGGCCTGAGCACAGGACAATTATGATGACCAACGCAACGGGCGAACGTAATCTGGCGCTGATCCAGGAAGTCCTGGAGGTGTTCCCGGAAACCGCGCGAAAAGAGCGCAGAAAGCACATGATGGTCAGCGATCCGGAAATGGAGAGCGTCGGCAAGTGCATTATCTCTAACCGCAAATCACAACCCGGCGTAATGACCGTACGCGGCTGCGCCTACGCCGGTTCCAAAGGGGTGGTATTTGGGCCGATTAAGGATATGGCCCATATTTCGCACGGACCGGTCGGCTGCGGCCAGTATTCCCGCGCCGGACGACGCAACTACTACACCGGAGTCAGCGGCGTCGATAGCTTCGGCACGCTGAACTTCACCTCTGATTTTCAGGAGCGCGACATCGTCTTCGGCGGCGATAAAAAGCTCAGCAAGCTGATTGAAGAGATGGAGTTGCTGTTCCCGCTCACCAAAGGGATCACCATTCAGTCGGAATGCCCGGTGGGGCTGATCGGTGATGATATCAGCGCGGTGGCCAACGCCAGCAGCAAGGCGCTGGATAAACCGGTGATCCCGGTACGCTGCGAAGGCTTTCGCGGCGTGTCGCAGTCTCTGGGGCACCATATCGCCAACGACGTGGTGCGCGACTGGATCCTGAACAATCGCGAAGGACAGCCGTTTGAAACCACCCCTTACGATGTGGCGATCATCGGCGACTACAACATCGGCGGCGACGCCTGGGCCTCGCGCATTCTGCTGGAAGAGATGGGGCTACGGGTAGTCGCGCAGTGGTCCGGCGACGGCACGCTGGTGGAGATGGAGAATACCCCATTCGTCAAGCTGAACCTGGTTCACTGCTACCGTTCGATGAACTATATCGCCCGCCATATGGAGGAGAAACATCAGATTCCGTGGATGGAGTACAACTTCTTCGGGCCGACCAAAATCGCCGAATCGCTGCGCAAAATCGCCGACCAGTTCGACGATACCATTCGCGCGAACGCCGAAGCGGTGATCGCCCGGTATGAGGGGCAGATGGCGGCGATTATCGCCAAATATCGCCCGCGCCTGGAGGGGCGTAAGGTGCTGCTCTATATGGGCGGCCTGCGGCCGCGCCACGTTATTGGCGCCTATGAGGATCTCGGGATGGAGATCATCGCCGCCGGCTACGAGTTTGCCCATAACGATGATTACGACCGCACCCTGCCGGATCTGAAAGAGGGCACGCTGCTGTTCGATGACGCCAGCAGCTACGAGCTGGAAGCGTTCGTCAAGGCGCTGAAGCCCGACCTTATCGGCTCCGGCATCAAGGAAAAATATATCTTCCAGAAAATGGGCGTGCCGTTCCGCCAGATGCACTCGTGGGACTATTCCGGCCCGTACCACGGCTACGATGGTTTCGCCATTTTCGCCCGCGATATGGATATGACCCTGAACAACCCGGCGTGGAACGAACTGACCGCTCCGTGGCTGAAGTCTGCGTGATTGCCCACTCACTGTCCCGTCTGTTCACCGATTTGTGGCGCGGGAGGAGAACACCATGAGCCAAACGATTGATAAAATTAATAGCTGTTATCCGCTATTCGAACAGGATGAATACCAGGAGCTGTTCCGCAATAAGCGGCAGCTGGAAGAGGCGCACGATGCGCAGCGCGTGCAGGAGGTCTTTGCCTGGACCACCACCGCCGAGTATGAAGCGCTGAATTTCCAGCGCGAGGCGCTGACCGTTGACCCGGCGAAAGCCTGCCAGCCGCTTGGCGCGGTGCTTTGCTCGCTGGGATTTGCCAACACCCTGCCGTATGTGCACGGCTCTCAGGGGTGCGTGGCCTACTTTCGCACCTATTTTAACCGCCATTTCAAAGAGCCGATCGCCTGCGTCTCCGACTCGATGACCGAAGACGCGGCGGTCTTCGGCGGCAACAACAATATGAACCTGGGCCTGCAGAACGCCAGCGCGCTGTACAAACCGGAGATCATTGCGGTGTCCACCACCTGCATGGCGGAAGTTATCGGCGATGACCTGCAGGCGTTTATCGCCAACGCTAAAAAAGATGGCTTCGTCGACAGCAGCATCGCCGTGCCCCACGCCCATACGCCAAGCTTTATCGGCAGCCACGTCACCGGCTGGGATAACATGTTTGAAGGCTTCGCCAAAACCTTCACTGCGGACTACCAGGGGCAGCCGGGCAAATTGCCGAAGCTCAATCTGGTGACCGGCTTTGAAACCTATCTCGGCAACTTCCGCGTATTAAAGCGGATGATGGAACAGATGGCGGTGCCGTGCAGCCTGCTCTCCGATCCGTCGGAAGTTCTCGACACGCCCGCCGACGGCCACTATCGGATGTATTCCGGCGGCACCACGCAGCAGGAGATGAAAGAGGCCCCTGACGCCATCGATACGCTGCTCCTGCAGCCGTGGCAGCTGCTGAAGAGCAAAAAAGTGGTGCAGGAGATGTGGAACCAGCCCGCCACCGAGGTCGCCATTCCGCTGGGGCTGGCCGCCACCGATGAACTGCTGATGACCGTCAGCCAGCTTAGCGGCAAGCCGATTGCCGACGCCCTCACCCTTGAGCGCGGCCGGCTGGTTGACATGATGCTCGACTCCCACACCTGGCTGCACGGCAAGAAGTTTGGCCTGTACGGCGATCCGGACTTCGTGATGGGCCTCACCCGCTTCCTGCTGGAGCTGGGCTGCGAGCCAACGGTGATCCTGAGCCATAACGCCAACAAACGCTGGCAAAAAGCGATGAACAAAATGCTCGATGCCTCGCCGTACGGGCGCGATAGCGAAGTGTTTATCAACTGCGATTTGTGGCACTTCCGTTCGCTGATGTTCACCCGTCAGCCGGACTTTATGATCGGCAACTCCTACGGCAAGTTTATCCAGCGCGATACCCTGGCGAAGGGTAAAGCCTTTGAAGTGCCGCTTATCCGCCTCGGCTTTCCGCTGTTCGACCGCCACCATCTGCACCGCCAGACAACCTGGGGTTATGAAGGGGCGATGAACATTGTGACGACGCTGGTGAACGCCGTGCTGGAGAAACTGGATAGCGATACCAGCCAGCTGGGCAAAACCGATTACAGCTTCGATCTCGTCCGTTAACCATCAGGTGCCCCGCGTCATGCGGGGCCAGGAGGGAGTATGCCCATCGTGATTTTCCGTGAGCGCGGCGCGGACCTGTACGCCTATATCGCGAAACAGGATCTGGAAGCGCGAGTGATCCAGATTGAGCATAACGACGCTGAACGCTGGGGCGGCGCGATTTCGCTGGAGGGGGGACGCCGCTACTACGTGCATCCGCAGCCGGGGCGTCCCGTCTTTCCGATAAGCCTGCGCGCGACGCGCAATACCTTGATATAAGGAGCTAGTGATGTCCGACAACGATACCCTATTCTGGCGTATGCTGGCGCTGTTTCAGTCTCTGCCGGACCTACAGCCGGCGCAAATCGTCGACTGGCTGGCGCAGGAGAGCGGCGAGACGCTGACGCCAGAGCGTCTGGCGACCCTGACCCAGCCGCAGCTGGCCGCCAGCTTTCCCTCCGCGACGGCGGTGATGTCCCCCGCTCGCTGGTCGCGGGTGATGGCGAGCCTGCAGGGCGCGCTGCCCGCCCATTTACGCATCGTTCGCCCTGCCCAGCGCACGCCGCAGCTGCTGGCGGCATTTTGCTCCCAGGATGGGCTGGTGATTAACGGCCATTTCGGCCAGGGACGACTGTTTTTTATCTACGCGTTCGATGAACAAGGCGGCTGGTTGTACGATCTGCGCCGCTATCCCTCCGCCCCCCACCAGCAGGAGGCCAACGAAGTGCGCGCCCGGCTTATTGAGGACTGTCAGCTGCTGTTTTGCCAGGAGATAGGCGGGCCCGCCGCCGCGCGGCTGATCCGCCATCGCATCCACCCGATGAAAGCGCAGCCCGGGACGACGATTCAGGCACAGTGCGAGGCGATCAATACGCTGCTGGCCGGCCGTTTGCCGCCGTGGCTGGCGAAGCGGCTTAACAGGGATAACCCTCTGGAAGAACGCGTTTTTTAATCCCTGTTTTGTGCTTGTTGCCCGCTGACCCCGCGGGCTTTTTTTCGCGTATGGACGCTCTTCCCCACGTTACGCTCAGGGGAATATTCCGTTCACGGTTGTTCCGGGCTTCTTGATGCGCCTAACCCCCTCGCTGCCAGCCTTTCATCAACAAATAGCCATCCCAGCGCGATAGGTCATAAAGCATCACATGCCGCCATCCCTTGTCCGATTGTTGGCTTTGTCGCAAAGCCAACAACCTCTTTTCTTTAAAAATCAAGGCTCCGCTTCTGGAGCGCGAATTGCATCTTCCCCCTCATCCCCCACCGTCAACGAGGTCACTATGAAGGGAAATGAAATTCTGGCGCTGCTGGATGAACCGGCCTGTGAACACAACCATAAACAAAAATCCGGCTGCAGCGCGCCCAAACCCGGCGCCACCGCCGGCGGCTGCGCGTTCGACGGCGCGCAGATAACCCTGCTGCCCATCGCCGACGTGGCGCATCTGGTCCACGGCCCCATCGGCTGCGCCGGAAGCTCATGGGATAACCGCGGCAGCGCCAGCTCCGGCCCCACCCTTAATCGGCTCGGGTTCACCACCGATCTCAACGAACAGGACGTGATTATGGGCCGCGGCGAACGCCGCTTGTTTCACGCCGTGCGCCATATCGTCACCCGCTATCATCCGGCGGCGGTCTTTATCTACAACACCTGCGTACCGGCCATGGAGGGCGATGACCTGGAAGCGGTATGCCAGGCCGCGCAGACCGCCACCGGCGTACCGGTTATCGCTATTGACGCCGCCGGTTTCTACGGCAGTAAAAATCTCGGTAACCGGCTGGCGGGCGACGTCATGGTCAAACGGGTCATCGGCCAGCGCGAGCCCGCCCCCTGGCCGGAGAGCACGCTCTTTGCCCCGGAGCAGCGTCACGATATTGGCCTGATTGGCGAATTCAATATTGCCGGCGAGTTCTGGCATATTCAGCCGCTGCTCGACGAACTGGGGATCCGCGTGCTCGGCAGCCTCTCCGGTGATGGCCGCTTCGCCGAGATCCAGACCATGCACCGGGCGCAGGCCAATATGCTGGTCTGCTCGCGGGCGTTAATTAACGTCGCCAGAGCCCTGGAGCAGCGCTACGGCACGCCGTGGTTCGAAGGCAGCTTTTACGGGATCCGCGCCACCTCTGACGCCCTGCGCCAGCTGGCGGCGCTGCTGGGCGACGACGACCTTCGCCAGCGCACCGAAGCGCTGATTGCGCGGGAGGAACAGGCGGCGGAACTGGCGCTACAGCCGTGGCGCGAACAGCTGCGCGGCCGCAAAGCGCTGCTCTATACCGGCGGGGTGAAATCCTGGTCGGTGGTATCGGCGCTGCAGGATTTGGGCATGACCGTGGTGGCAACCGGCACGCGTAAATCCACCGAAGAGGATAAACAGCGGATCCGCGAGCTGATGGGCGAAGAGGCGGTAATGCTGGAAGAGGGCAACGCCCGCACGCTgctggatgtggtctATCGCTATCAGGCCGACCTGATGATTGCCGGCGGACGCAATATGTACACCGCCTATAAAGCCAGGCTGCCGTTTCTCGATATCAATCAGGAGCGCGAACACGCCTTCGCTGGCTATCAGGGGATCGTCACCCTCGCCCGCCAGCTGTGTCAGACCATCAACAGCCCCATCTGGCCGCAAACCCATTCTCGCGCCCCGTGGCGCTAAGGAGCTCACCATGGCAGACATTTTCCGCACCGATAAGCCGCTGGCGGTCAGCCCCATCAAAACCGGCCAGCCGCTCGGCGCAATCCTCGCCAGCCTCGGGATCGAACACAGCATCCCTCTGGTCCACGGCGCGCAGGGGTGCAGCGCCTTCGCCAAAGTCTTTTTTATTCAACATTTCCACGACCCGGTTCCCCTGCAGTCGACGGCGATGGACCCCACGTCGACGATTATGGGCGCGGACGGCAATATTTTTACCGCCCTGGATACCCTCTGCCAGCGCAACAATCCGCAGGCTATCGTACTGCTCAGCACCGGGCTGTCGGAGGCCCAGGGCAGCGATATTTCCCGCGTGGTTCGCCAGTTTCGCGAAGAGTATCCCCGGCATAAGGGGGTGGCGATATTGACGGTTAACACGCCGGATTTTTATGGCTCCATGGAGAACGGCTTCAGCGCGGTGTTAGAGAGCGTCATTGAGCAGTGGGTGCCGCCGGCGCCGCGCCCGGCTCAGCGCAATCGCCGGGTCAATCTGCTGGTCAGCCATCTCTGTTCGCCGGGCGATATCGAGTGGCTGCGCCGATGCGTCGAAGCCTTTGGTCTGCAGCCGATAATCCTGCCGGACCTGGCGCAATCGATGGACGGCCACCTGGCGCAGGGCGATTTCTCGCCGCTGACCCAGGGCGGGACGCCGCTGCGCCAGATAGAGCAGATGGGGCAAAGCCTGTGCAGCTTCGCCATTGGCGTCTCCCTTCATCGCGCCTCATCGCTGCTGGCCCCGCGCTGCCGCGGCGAGGTTATCGCCCTGCCGCACCTGATGACCCTCGAACGCTGCGACGCCTTTATTCATCAACTGGCGAAAATTTCCGGACGCGCCGTTCCCGAGTGGCTGGAACGCCAGCGCGGCCAGCTACAGGATGCGATGATCGACTGCCATATGTGGCTCCAGGGCCAGCGCATGGCGATAGCGGCGGAAGGCGATTTGCTGGCGGCGTGGTGTGATTTCGCCAACAGCCAGGGGATGCAGCCCGGCCCGCTGGTGGCCCCTACCGGTCATCCCAGCCTGCGCCAGCTGCCGGTGGAACGGGTGGTGCCGGGGGATCTGGAGGATCTGCAAACCCTGCTGTGCGCGCATCCCGCCGACCTGCTGGTGGCGAACTCGCACGCCCGCGACCTGGCGGAGCAGTTTGCGCTGCCGCTGGTGCGCGCGGGTTTTCCGCTCTTTGACAAGCTCGGCGAATTCCGCCGGGTGCGACAGGGGTATAGCGGGATGCGCGATACGCTGTTTGAGCTGGCAAACCTGATACGCGAGCGTCACCACCACCTCGCCCACTACCGATCGCCGCTGCGCCAGAACCCCGAATCGTCACTCTCCACAGGAGGCGCTTATGCCGCCGATTAACCGTCAGTTTGATATGGTCCACTCCGATGAGTGGTCTATGAAGGTCGCCTTCGCCAGCTCCGACTATCGTCACGTCGATCAGCACTTCGGCGCTACCCCGCGGCTGGTGGTGTACGGCGTCAAGGCGGATCGGGTCACTCTCATCCGGGTGGTTGATTTCTCGGTCGAGAACGGCCACCAGACGGAGAAGATCGCCAGGCGGATCCACGCCCTGGAGGATTGCGTCACGCTGTTCTGCGTGGCGATTGGCGACGCGGTTTTTCGCCAGCTGTTGCAGGTGGGCGTGCGTGCCGAACGCGTTCCCGCCGACACCACCATCGTCGGCTTACTGCAGGAGATTCAGCTCTACTGGTACGACAAAGGGCAGCGCAAAAATACGCGCCAGCGCGACCCGGAGCGCTTTACCCGTCTGCTGCAGGAGCAGGAGTGGCATGGGGATCCGGACCCGCGCCGCTAGCCGTGTCGTTTCTGTGACAAAGCCCACAAAACATCGCGACACTGTAGGACGAACCTTGTCAGGACTAATACACAACCATTTGAAAAATATTAATTTTATTCTCTGGTATCGCAATTGCTAGTTCGTTATCGCCACCGCGCTTCCGCGGTGAACCGCGCCCCGGCGTTTTCCGTCAACATCCCTGGAGCTGACAGCATGTGGAATTACTCCGAGAAAGTGAAAGACCATTTTTTTAACCCCCGCAATGCGCGCGTGGTGGACAACGCCAACGCGGTAGGCGACGTCGGTTCGTTAAGCTGCGGCGACGCCCTGCGCCTGATGCTGCGCGTCGACCCGCAAAGCGAAATCATTGAGGAGGCGGGCTTccagaccttcggctgCGGCAGCGCCATCGCCTCCTCCTCCGCGCTGACGGAGCTGATTATCGGCCATACCCTCGCCGAAGCCGGGCAGATAACCAATCAGCAGATTGCCGATTATCTCGACGGACTGCCGCCGGAGAAAATGCACTGCTCGGTGATGGGCCAGGAGGCCCTGCGCGCGGCCATCGCCAACTTTCGCGGCGAAAGCCTTGAAGAGGAGCACGACGAGGGCAAGCTGATCTGCAAATGCTTCGGCGTCGATGAAGGGCATATTCGCCGCGCGGTACAGAACAACGGGCTGACCACCCTTGCCGAGGTGATCAACTACACCAAAGCGGGCGGCGGCTGCACCTCTTGCCACGAAAAAATCGAGCTGGCCCTGGCGGAGATCCTCGCCCAGCAGCCGCAGACGACGCCAGCCGTGGCCAGCGGCAAAGATCCGCACTGGCAGAGCGTCGTCGATACCATCGCAGAACTGCGGCCGCATATTCAGGCCGACGGCGGCGATATGGCGCTACTCAGCGTCACCAACCACCAGGTGACCGTCAGCCTCTCCGGCAGCTGTAGCGGCTGCATGATGACCGATATGACCCTGGCCTGGCTGCAGCAAAAACTGATGGAACGTACCGGCTGTTATATGGAAGTGGTGGCGGCCTGAGCCGCGGTTAACTGACCCAAGGGGGACAAGATGAAACAGGTTTATCTCGATAACAACGCCACCACCCGTCTGGACCCGATGGTCCTGGAAGCGATGATGCCCTTTTTGACCGATTTTTACGGCAACCCCTCGTCGATACACGATTTTGGCATTCCGGCCCAGGCGGCTCTGGAACGCGCGCATCAGCAGGCTGCGGCGCTGCTGGGCGCGGAGTATCCCAGCGAGATCATCTTTACCTCCTGCGCCACCGAAGCCACCGCCACCGCCATCGCCTCGGCGATCGCCCTGCTGCCTGAGCGTCGCGAAATCATCACCAGCGTGGTCGAACATCCGGCGACGCTGGCGGCCTGCGAGCACCTGGAGCGCCAGGGCTACCGGATTCATCGCATCGCGGTGGATAGCGAGGGGGCGCTGGACATGGCGCAGTTCCGCGCGGCGCTCAGCCCGCGCGTCGCGTTGGTCAGCGTGATGTGGGCGAATAACGAAACCGGGGTGCTTTTCCCGATCGGCGAAATGGCGGAGCTGGCCCATGAACAAGGGGCGCTGTTTCACTGCGATGCGGTGCAGGTGGTCGGGAAAATACCGATCGCCGTGGGCCAGACCCGCATCGATATGCTCTCCTGCTCGGCGCATAAGTTCCACGGGCCAAAAGGCGTAGGCTGTCTTTATCTGCGGCGGGGAACGCGCTTTCGCCCGCTGCTGCGCGGCGGTCACCAGGAGTACGGTCGGCGAGCCGGGACAGAAAATATCTGCGGAATCGTCGGCATGGGCGCGGCCTGCGAGCTGGCGAATATTCATCTGCCGGGAATGACGCATATCGGCCAATTGCGCAACAGGCTGGAGCATCGCCTGCTGGCCAGCGTGCCGTCGGTCATGGTGATGGGCGGCGGCCAGCCGCGGGTGCCCGGCACGGTGAATCTGGCCTTTGAGTTTATTGAAGGTGAAGCCATTCTGCTGCTGTTAAACCAGGCCGGGATCGCCGCCTCCAGCGGCAGCGCCTGCACCTCAGGCTCGCTGGAACCCTCCCACGTGATGCGGGCGATGAATATCCCCTACACCGCCGCCCACGGCACCATCCGCTTTTCTCTCTCGCGCTACACCCGGGAGAAAGAGATCGATTACGTCGTCGCCACGCTGCCGCCGATTATCGACCGGCTGCGCGCGCTGTCGCCCTACTGGCAGAACGGCAAGCCGCGCCCGGCGGACGCCGTATTCACGCCGGTTTACGGCTAAGGCGGAGGTGGCTGATGGAACGCGTGCTGATTAACGATACCACCCTGCGCGACGGCGAGCAGAGCCCCGGCGTCGCCTTTCGCACCAGCGAAAAGGTCGCCATTGCCGAGGCGCTTTACGCCGCAGGAATAACGGCGATGGAGGTCGGCACCCCGGCGATGGGCGACGAGGAGATCGCGCGGATCCAGCTGGTGCGTCGCCAGCTGCCCGACGCGACCCTGATGACCTGGTGTCGGATGAACGCGCTGGAGATCCGCCAGAGCGCCGATCTGGGCATCGACTGGGTGGATATCTCGATTCCGGCTTCGGATAAGCTGCGGCAGTACAAACTGCGCGAGCCGCTGGCGGTGCTGCTGGAGCGGCTGGCGATGTTTATCCATCTTGCGCATACCCTCGGCCTGAAGGTATGCATCGGCTGCGAGGACGCCTCGCGGGCCAGCGGCCAGACCCTGCGCGCTATCGCCGAGGTCGCGCAGCAATGCGCCGCCGCCCGCCTGCGCTATGCCGATACGGTCGGCCTGCTCGACCCTTTTACCACCGCGGCGCAAATCTCGGCCCTGCGCGACGTCTGGTCCGGCGAAATCGAAATGCATGCCCATAACGATCTGGGTATGGCGACCGCCAATACGCTGGCGGCGGTAAGCGCCGGGGCCACCAGCGTGAATACGACGGTCCTCGGTCTCGGCGAGCGGGCGGGCAACGCGGCGCTGGAAACCGTCGCGCTGGGCCTTGAACGCTGCCTGGGCGTGGAGACCGGCGTGCATTTTTCGGCGCTGCCCGCGCTCTGTCAGAGGGTCGCGGAAGCCGCGCAGCGCGCCATCGACCCGCAGCAGCCGCTGGTCGGCGAGCTGGTGTTTACCCATGAGTCAGGTGTCCACGTGGCGGCGCTGCTGCGCGACAGCGAGAGCTACCAGTCCATCGCCCCTTCCCTGATGGGCCGCAGCTACCGGCTGGTGCTGGGCAAACACTCCGGGCGTCAGGCGGTCAACGGCGTTTTTGACCAGATGGGCTATCACCTCAACGCCGCGCAGATTAACCAGCTGCTGCCCGCCATCCGCCGCTTCGCCGAGAACTGGAAGCGCAGCCCGAAAGATTACGAGCTGGTGGCTATCTACGACGAGCTGTGCGGTGAATCCGCTCTGCGGGCGAGGGGGTAATGATGGAGTGGTTTTATCAAATTCCCGGCGTGGACGAACTTCGCTCCGCCGAATCTTTTTTTCAGTTTTTCGCCGTCCCCTATCAGCCCGAGCTGCTTGGCCGCTGCAGCCTGCCGGTGCTGGCAACGTTTCATCGCAAACTCCGCGCGGAGGTGCCGCTGCAAAACCGGCTCGAGGATAACGACCGCGCGCCCTGGCTGCTGGCGCGAAGACTGCTCGCGGAGAGCTATCAGCAACAGTTTCAGGAGAGCGGAACATGAGACCGAAATTCACCTTTAGCGAAGAGGTCCGCGTCGTACGCGCGATTCGTAACGACGGCACCGTGGCGGGCTTCGCGCCCGGCGCGCTGCTGGTCAGGCGCGGCAGCACCGGCTTTGTGCGCGACTGGGGCGTTTTTTTGCAAGATCAGATTATCTACCAGATCCACTTTCCGGAAACCGATCGGATCATCGGCTGCCGCGAGCAGGAGCTGATCCCCATCACCCAGCCGTGGCTGGCCGGAAATTTGCAATACAGGGATAGCGTGACCTGCCAGATGGCGCTCGCGGTCAACGGCGATGTGGTCGTGAGCGCCGGCCAGCGGGGACGCGTTGAGGCTACCGATCGGGGANAGCTCGGCGACAGCTACACCGTCGACTTTAGCGGCCGCTGGTTCAGGGTCCCGGTGCAGGCCATCGCCCTTATAGAGGAAAGAGAAGAATGAACCCATGGCAACGTTTTGCCCGGCAGCGGCTGGCGCGCAGCCGCTGGAATCGCGATCCGGCGGCCCTGGATCCGGCCGATACGCCGGCTTTTGAACAGGCCTGGCAACGCCAGTGCCATATGGAGCAGACGATCGTCGCGCGGGTCCCTGAAGGCGATATTCCGGCGGCGTTGCTGGAGAATATCGCTGCCTCCCTTGCCATCTGGCTCGACGAGGGGGATTTTGCGCCGCCCGAGCGCGCTGCCATCGTGCGCCATCACGCCCGGCTGGAACTCGCCTTCGCCGATATCGCCCGCCAGGCGCCGCAGCCGGATCTCTCCACGGTACAGGCATGGTATCTGCGCCACCAGACGCAGTTTATGCGCCCGGAACAGCGTCTGACCCGCCATTTACTGCTGACGGTCGATAACGACCGCGAAGCCGTGCACCAGCGGATCCTCGGCCTGTATCGGCAAATCAACGCCTCGCGGGACGCTTTCGCGCCGCTGGCCCAGCGCCATTCCCACTGCCCGAGCGCGCTGGAAGAGGGTCGTTTAGGCTGGATTAGCCGTGGCCTGCTCTATCCGCAGCTCGAGACCGCGCTGTTTTCACTGGCGGAAAACGCGCTAAGCCTTCCCATCGCCAGCGAACTGGGCTGGCATCTTTTATGGTGCGAAGCGATTCGCCCCGCCGCGCCCATGGAGCCGCAGCAGGCGCTGGAGAGCGCGCGCGATTATCTTTGGCAGCAGAGCCAGCAGCGCCATCAGCGCCAGTGGCTGGAACAGATGATTTCCCGTCAGCCGGGACTGTGCGGGTAGCCTCGGCGGCTACCCGTTAACGCCTACAGCACGGTGCGTTTAATCTCCTCAAGCCAGCTCGCCAGACGCGCTTCGGTCTGGTCGAACTGGTTATCCTGATCCAGCACCAGCCCAACAAAGCGGTCGCCTTCCAGCGCCGAGGACGCGCTGAATTCATAACCCTCATTTGGCCAGCTGCCAATCATCTGCGCGCCGCGCGCGCTCAGGGCGTCGAACAGCGGGCGCATCCCGCTGACGAAGTTGTCCGGATAGCCTCTCTGATCGCCGAGGCCGAACAGCGCCACGGTTTTCCCTTTCAGGCTGGCGTCGTCGAGGCCGCTGATAAATTCGCTCCATGACTCGCTTTCGCATCCGGCCTCCAGCCCCGGCAGCTGGCCGTCGCCGAGCGTCGGCGTGCCCAGCAGCAGCACCGGATAGGCCATAAAGTCGTCCAGCGTCGTGCGGTTAATGTTGACCGGGGCATCCGCCAGCTCGCCCAGTTGCTTATGGATCATTTTCGCGATTTTGCGGGTTTTACCGGTATCGGTGCCAAAGAAAATACCAATGTTCGCCATGTTGCGCTCCTGTCGGAAAAGGGGGTTGAAAATACGCGTTCTCGCAGGGGTATTGCGAAGGCTGTGCCAGGTTGCTTTGCACTACCGCGGCCCATCCCTGCCCCAAAACGATCGCTTCAGCCCTCTCCCGCCGCGCGCGGCGGGGCTGGCGGGGCGCTTAAAATGCAAAAAGCGCCTGCTTTTCCCCTACCGGATCAATGTTTCTGCACATCACGCCGATAAGGGCGCACGGTTTGCATGGTTATCACCGTTCGGAAAACACCGCGGCGTCCCTGTCACGGTGTCGGACAAATTGTCATAACTGCGACACAGGAGTTTGCGATGACCCTGAATATGATGCTCGATAACGCCGTACCCGAGGCGATTGCCGGTGCGCTGACTCAACAACATCCGGGGCTGTTTTTTACAATGGTCGAACAGGCATCGGTAGCGATTTCCCTCACCGATGCCCGGGCGAATATTATCTACGCCAACCCGGCGTTTTGCCGCCAGACTGGATACTCGCTGGCGCAATTGCTCAATCAAAACCCGCGCCTGCTGGCCAGCAGCCAGACGCCGCGCGAGATCTACCAGGAGATGTGGCAAACCCTGCTCCAGCGCCAGCCGTGGCGCGGTCAGCTAATTAATCAGCGCCGCGACGGCGGCCTGTATCTGGTAGATATCGATATCACGCCGGTGCTGAATCCGCAGGGCGAGCTGGAGCATTATCTGGCGATGCAGCGGGATATCAGCGTCAGCTATACCCTGGAACAGCGGCTGCGCAATCATATGACGCTAATGGAAGCGGTGCTCAATAACATCCCCGCCGCCGTGGTCGTGGTCGATGAGCAGGATCGGGTGGTGATGGATAATCTCGCCTACAAAACGTTCTGCGCGGACTGCGGCGGGAAAGAGCTGCTGGTCGAGCTCCAGGTTTCCCCGCGCAAAATGGGGCCCGGCGCGGAGCAAATCCTGCCGGTGGTGGTTCGCGGCGCGGTCCGCTGGCTGTCGGTAACCTGCTGGGCGCTGCCCGGCGTGAGTGAAGAAGCCAGCCGCTACTTCGTCGACAGCGCCCCGGCGCGCACGCTGATGGTGATCGCCGACTGTACCCAGCAGCGCCAGCAGCAGGAGCAGGGCCGGCTCGACCGTCTGAAACAGCAAATGACCGCCGGTAAGCTGCTGGCCGCGATTCGCGAGTCGCTGGACGCGGCGCTGATTCAGCTTAATTGCCCAATCAATATGCTGGCGGCGGCCCGCCGGCTGAACGGCGAAGGCAGCGGCAACGTGGCGCTGGACGCGGCGTGGCGCGAAGGTGAAGAGGCCATGGCGCGCCTGCAGCGCTGCCGCCCTTCTCTTGAGCTGGAAAGCAATGCCGTCTGGCCGCTTCAGCCCTTTTTTGACGACCTGTACGCCCTCTACCGCACCCGCTTTGACGATCGCGCGCGGCTGCAGGTGGACATGGCATCGCCGCATCTGGTCGGCTTCGGCCAGCGTACCCAGCTGCTGGCCTGCTTGAGTTTATGGCTCGACCGGACGCTGGCCCTCGCCGCCGAGCTGCCCTCCGTACCGCTGGAGATCGAGCTTTACGCCGAAGAGGACGAGGGCTGGCTCTCTTTGTATCTCAACGACAATGTCCCGCTGCTGCAGGTGCGCTACGCCCACTCCCCCGATGCCCTAAACTCTCCCGGCAAAGGGATGGAGCTGCGGCTGATCCAAACGCTGGTCGCCTACCACCGCGGCGCGATTGAACTGGCTTCGCGACCGCAGGGAGGCACCAGCCTGGTTCTGCGTTTCCCGCTCTTTAATACCCTGACCGGAGGTGAGCAATGATCCATAAATCCGATTCGGACACCACCGTCAGACGTTTCGATCTCTCCCAGCAGTTTACCGCCATGCAGCGGATAAGCGTGGTCCTGAGTCGCGCCACCGAAGCGAGCAAAACCCTGCAGGAGGTTCTGAGCGTGCTACATAACGATGCCTTTATGCAGCACGGGATGATTTGCCTGTACGACAGCCAGCAGGAGATCCTGAGCATCGAAGCGCTGCAGCAAACGGAAGATCAGACGCTGCCCGGCAGTACGCAAATTCGCTACCGGCCGGGGGAAGGATTAGTCGGTACCGTGCTGGCGCAGGGCCAGTCGCTGGTGCTGCCGCGCGTCGCCGACGACCAGCGTTTTCTCGATCGTCTGAGCCTGTACGACTATGACCTGCCGTTTATCGCCGTTCCGCTGATGGGCCCCCACTCCCGGCCCATCGGCGTACTGGCGGCGCAGCCGATGGCGCGTCAGGAAGAGCGGCTGCCCGCCTGCACGCGCTTTCTCGAAACCGTCGCCAATCTGATCGCCCAGACGATTCGCCTGATGATCCTGCCAACCTCCGCCGCGCAGGCGCCGCAGCAGAGCCCCAGAATAGAGCGCCCGCGCGCCTGTACCCCTTCGCGCGGTTTCGGCCTGGAAAATATGGTCGGTAAAAGCCCGGCGATGCGGCAGATTATGGATATTATTCGTCAGGTTTCCCGCTGGGATACCACGGTGCTGGTACGCGGCGAGAGCGGCACCGGGAAAGAGCTCATCGCCAACGCCATCCACCATAATTCTCCGCGCGCCGCCGCGGCGTTCGTCAAATTTAACTGCGCGGCGCTGCCGGACAACCTGCTGGAGAGCGAGCTGTTTGGTCATGAGAAAGGCGCGTTTACCGGCGCGGTGCGCCAGCGGAAAGGCCGCTTTGAGCTGGCGGACGGCGGCACCTTATTCCTCGATGAGATCGGCGAAAGCAGCGCCTCGTTTCAGGCTAAGCTACTGCGTATTCTGCAAGAGGGGGAGATGGAGCGCGTCGGCGGCGACGAAACCCTGCGGGTCAACGTGCGCATTATCGCGGCGACCAACCGCCATCTGGAAGAGGAGGTGCGGCTGGGTCATTTCCGCGAGGATCTATACTACCGCCTGAACGTAATGCCTATCGCGCTGCCGCCGCTGCGCGAGCGCCAGGAGGATATCGCCGAGCTGGCGCACTTTCTGGTGCGAAAAATCGCCCACAGCCAGGGGCGAACGCTGCGCATCAGCGATGGGGCGATTCGCCTGCTGATGGAGTACAGCTGGCCGGGAAACGTGCGCGAACTGGAAAACTGTCTCGAACGTTCGGCGGTGCTGTCGGAAAGCGGCCTGATAGACCGGGACGTGATTCTGTTCAACCATCGCGATAACCCGCCGAAAGCGCTCGCCAGCAGCGGCCCGGCGGAGGACGGCTGGCTCGATAACAGCCTCGACGAGCGCCAGCGGCTGATCGCCGCCCTGGAAAAAGCGGGCTGGGTGCAGGCCAAAGCGGCGCGGCTGCTCGGCATGACCCCGCGCCAGGTGGCGTATCGCATTCAGATTATGGATATCACCATGCCGCGACTGTGAAGCCTTATGTGAGATTCAGGACATTGTCGCCAGCGCGGCGGAATTGCGACAATTCAGGGACGCGGGTTGCCGGTTAAAAAGTCTACTTTTCATGCGGTTGCGAAATTAACCTCTGGTACAGCATTTGCAGCAGGAAGGTATCGCCCAACCACGAAGGTACGACCATGACTTCCTGCTCCTCTTTTTCTGGCGGCAAAGCCTGCCGCCCGGCGGATGACAGCGCATTGACGCCGCTTGTGGCCGATAAAGCTGCCGCGCACCCCTGCTACTCTCGCCATGGGCATCACCGTTTCGCGCGGATGCATCTGCCCGTCGCGCCCGCCTGCAATTTGCAGTGCAACTACTGTAATCGCAAATTCGATTGCAGCAACGAGTCCCGCCCCGGGGTATCGTCAACGCTGCTGACGCCTGAACAGGCGGTCGTGAAAGTGCGTCAGGTCGCGCAGGCGATCCCGCAGCTTTCGGTGGTGGGCATCGCCGGGCCCGGCGATCCGCTCGCCAATATCGCCCGCACCTTTCGCACCCTGGAGCTGATCCGCGAACAGCTGCCGGACCTGAAATTATGCCTGTCGACCAACGGACTGATGCTGCCTGACGCGGTGGACCGCCTGCTGGATGTCGGCGTTGACCACGTCACGGTCACCATTAACACCCTCGACGCGGAGATTGCCGCGCAAATCTACGCCTGGCTATGGCTGGACGGCGAACGCTACAGCGGGCGCGAAGCGGGAGAGATCCTGATTGCCCGTCAGCTTGAGGGCGTACGCAGGCTGACCGCCAAAGGCGTGCTGGTGAAAATAAATTCGGTGCTGATCCCCGGTATCAACGATAGCGGCATGGCCGACGTGAGCCGCGCGCTGCGGGCCAGCGGCGCGTTTATCCATAATATTATGCCGCTGATCGCCAGGCCGGAGCACGGCACGGTGTTTGGCCTCAACGGCCAGCCGGAGCCGGACGCCGAGACGCTCGCCGCCACCCGCAGCCGGTGCGGCGAAGTGATGCCGCAGATGACCCACTGCCACCAGTGTCGCGCCGACGCCATTGGGATGCTCGGCGAAGACCGCAGCCAGCAGTTTACCCAGCTTCCGGCGCCAGAGAGTCTCCCGGCCTGGCTGCCGATCCTCCACCAGCGCGCGCAGCTGCACGCCAGCATTGCGACCCGCGGCGAATCTGAAGCCGATGACGCCTGCCTGGTCGCCGTGGCGTCAAGCCGCGGGGACGTCATTGATTGTCACTTTGGTCACGCCGACCGGTTCTACATTTACAGCCTCTCGGCCGCCGGTATGGTGCTGGTCAACGAGCGCTTTACGCCCAAATATTGTCAGGGGCGCGATGACTGCGAGCCGCAGGATAACGCAGCCCGGTTTGCGGCGATCCTCGAACTGCTGGCGGACGTTAAAGCCGTATTCTGCGTGCGTATCGGCCATACGCCGTGGCAACAGCTGGAACAGGAAGGCATTGAACCCTGCGTTGACGGCGCGTGGCGGCCGGTCTCCGAAGTGCTGCCCGCGTGGTGGCAACAGCGTCGGGGGAGCTGGCCTGCCGCGTTGCCGCATAAGGGGGTCGCCTGATGCCGCCGCTCGACTGGTTGCGGCGCTTATGGCTGCTGTACCACGCGGGGAAAGGCAGCTTTCCGCTGCGCATGGGGCTTAGCCCGCGCGATTGGCAGGCGCTGCGGCGGCGCCTGGGCGAGGTGGAAACGCCGCTCGACGGCGAGACGCTCACCCGTCGCCGCCTGATGGCGGAGCTCAACGCCACCCGCGAAGAGGAGCGCCAGCAGCTGGGCGCCTGGCTGGCGGGCTGGATGCAGCAGGATGCCGGGCCGATGGCGCAGATTATCGCCGAGGTTTCGCTGGCGTTTAACCATCTCTGGCAGGATCTTGGTCTGGCATCGCGCGCCGAATTGCGCCTGCTGATGAGCGACTGCTTTCCACAGCTGGTGGTGATGAACGAACACAATATGCGCTGGAAAAAGTTCTTTTATCGTCAGCGCTGTTTGCTGCAACAGGGGGAAGTTATCTGCCGTTCGCCAAGCTGCGACGAGTGCTGGGAACGCAGCGCCTGTTTTGAGTAGCCGTTTCCCGAAGGGGGCGCTGCAAACAAAAAAGCCGGAGGTTTCCCTCCGGCTTTTCACATCATCAAATGTGATTATGCGACGTCTTCGTACTGCGGCACCGGGTTGCGGAAGCTTTTGGTCACPseudomonas stutzeri A1501 nif cluster (SEQ ID NO. 5)gttaggttggcctgaattcggtgtgtatcccccggagatcagcttcgcctcggcacgctcagcctgcactcgccccagcctagctttccgccgcaagtgcggcatcgagtcgcgccaccaggctgccgtcggcttccaggccgaggatgatgtcgcaaccgccgaccagctcgccacgcaggaacagctgcgggtaggtcggccactgcgagatcttcggcagcttctcgcggatatgcggtgccagtagcacgttgaccgtggcgaacggccggccgctgttcttcaatgcctccaccgcggcgcgggagaaaccgcactccggcacgcccggcgtgcccttcatgtacagcagcaccggatgctcggcgagttgctggcgtatgcgtgcttcggtatcgagaacttgcatgcgttcactccattgccagggtgcagggggagttgtaggcgcaggggctggcatgggcccgctgtgggcgatccttccaggcctcgtagccgccgtccaggctgtagcaattgatgaagccgaaatcgctgaacagctgtgccatgtcacggctggcatgaccgtgctcgcaacagatgatcagatggacgtgatttggcgtgctcttgagcagcgtgcgcaagttcagctcgctgaggcgggtggcgcgcgggtcatggccctggcagtaggcgcgggcatcgcgcatgtccagcagcatggtgttttcggtcgccaacagccgctgggcctgctcgacgctgatgcgttggtagtcgctcattgctcttctccaaaacaatcgtgataggtcgggcaggcttcacaagagggggagcggcatacgtagccgccgtcctgttcgcatagttgtttgtagaggaacttcttccagcgcatgtcctgggtattgcgccgggccagctgcgggaagttgtgcatcagcagcgcgtacagctgcgcccgcgaggccaggccgaggtcgcgccacaggtgttcgccaccgaggcaggcggcagcgacgatggctgccatcgccggttcgccgtggtcgtcctggccgcccagcagcaggtcgtgcagcgcctgccattcttcccggcgcagcgccagcaactcttcgcgcaaggcgtcgcgctcgccgagcaaggcctcgtcggcgctacgggatggtggtcgcagcccatggcgcgtcaggagctcggcgtactgcgccgcgtcgagcccgaggtgctgcggcaggcaactacggccttcgcgctgggcgcggatgatctgcgctagccaggccgggttgtcgttgactgcgacctccaggcacaacgcggcccggctcattgcggcgtgctcgcggcggggctgcaaccgagcaccgagctggtgcccagcgaacagccgctgatgctcggtgccagcgctggcagccggcctttgcacggcagccaggcattgatgccgaccaggtcgaagtcgaccatgtagtgcatgccgcagcggatcagcgggcggcggatcaacagcggctgggccaccatcagctccagtgcctgttcggcgctcagttcgctgacatccagctcgccgtacttgatcgccggggccgacgggttgaaccactcggccaccggcagccggccgaagaacggccgcaggcgttccggcgtccaggcctcgcgcagcaggtcgcgcacttccagctcgatgcccgctgaacgcagcagctccttctgcaggcggttggtggcgcaaccgggcttctcgtagaagatgatgcaggacatggcaacctcctcaacgggcctggatggcgcgcatggcctcggccaaccgctccggcggaatgcccgtcagcgagccggctgggttggccgggctgccgtcggcgagcaggatcgcgccttcgatggggcagatgctcgcgcactgttgctcggcgtagtcgccatcgcattcggtgcacttgtgcgcgctgatgcggaagtacgcagtgccggggctgatcgcctcgctcgggcagacgtccacgcaggcccagcagttgacgcaggattcgacgatttgcagtgccatactccacctcctcatgccatcaggcattgctccgctgcgcccaccgacgcatcgagacggccattggcgatcatttcctggtacacctccagcacggcttcctcgatgggctccatggcgtgctcgccattgggctggatgccggcggcttccagctcgccccagggttcgaagccgatcttcgagcagagcaccgcctcgcagcccttgagcgcgcggatgctgcccgacagcgcactgtccttgtcgccgcagctgtcgttgccgacgcagtactgctcgaccttgcggtggccgatgaagcgcaccccggccggcgaggcctcgtagacgaggaattcgcgggcatggccgaagtgctggttgaccaggccgccgccgctggtggccacggccatcagtaccgggcgatggcccttgtccactgtgccggtgagctgcgcagcgctgggggtggccaggcgcgccttcttcgccgcgcgttcgtccagctcctccttgatcgccgcgtggatggcggcgcgcttgaccatcgccgcctcgtagtcgacgtccatgctctcgatcttgtcgagggtgaactcgtcgccgcggtcctcgccgagcaggcccaccgcgtcggcgcggcactggcggcagtggcgcatcatgttcatgtcgccggcacaggcgtcctgcaggtcctgcagttcctccggctccgggctgcgctggcccatcacgccatagaaggtgccgtgctcggcctcggcgatcagcggcatgacgttgtgcaggaaggcgcccttggccttgacgatgcggctgacctctttcaggtgctcatcgttgacgccggggatcagcaccgagttgaccttcaccaggatgccacgctcgaccagcatctccaggcccttctgctgccgttcgatgaggatcttggccgccttgcgcccacggatgcgcttgttgttccagtagatccaggggtagatctcggcgccgatgtccgggtccacgcagttgatggtgatggtcacgtggtcgatgttgtgcttggccagctcgtcgacgcagtcgggcagggccaggccgttggtggagacgcacagcttgatgtccggcgcctgctcggacagcatgcgaaaggtctcgaaggtgcgctgcgggttggccagcgggtcgcccgggccggcgatgccgagcacggtcatctgcgggatggtcgccgccaccgccttgaccttcttcaccgcttgcaccggctccagcagctcggacaccacgcccgggcgcgattcgttggcgcagtcgtacttgcggttgcagtagtggcactggatgttgcaggccggcgccaccgccacatgcatgcgcgcgaagtagtggtgcgcctcctcggagtagcaggggtggttgtgcactttctcgcggatgtgctcgggcaggtgcgcgagctggtcatccgagctgccacaggaacccgctgaacaaccgcccccggcggttggcccggcctcgctctggcccagtacgttcagttccatgttcggtctccgaatagaggtctgtccccggtacctgcagcaaggcttgtgcctgttttcaaatcattgtttcagaacgaatttttcagaaagcgggcggaattcgttgtttcgcaacgaacaaagtggcggggccgggcggggcggctgtcgcaaaggcgacaagctgcgcacgcccggttcccgggctgtcgcgacccggtgctccagacgattgcgcatggcgggccgcgatccgcaccagcgccccggcccgctggtgccgggctactcctcgaggcgcccgctggcgtcgcgatcgcgcacgtaatggtgggtgagcggaaacgccggcagccaggactcgcggcggacggtccagagctcgtaggtgggcatcagctggtcgggggcatccagggctcccaggctcacttcgatttcgtccgcggtgcgtgccctgctagtgatgcgtagtgggcacaaggcttcgcgggaagcgccatgcatggggaacgctgcgccgcccgacccgggagtcgggcgggtcgttcagatcttgcgcatatgaatgttcagcgtctgcactcggtaggcgatctgccggggcgtcatgccgagcaggcgggcggccttggcctggacccagccggcctgttccagcgcggcgatgacgcgctcgcggtcgtcgaggctgtcgtcggcgaggtcgacttcggggaccggcgccagcggcgtggcgtcgtggtcgaggccggtgagggagaccacgtcgcggctgatggtgccatcctcgctcatgatggccgagcgttccaggcagttttccagttcgcgcacgttgcccggccagcggtggctcatcagcagacgcagggcgctgtcggtcagcttgagtttgcgaccctgctggcgggcgatcttgtcgaggaggaattcggccagttccgggatgtcggcgctgcgctcgcgcagcggcgggacgcggatggccatgacgttgaggcggtagtagaggtcttcgcggaacttgccttgctccacctcgtgctccaggtcgcggttggtggcggcgacgatgcgcacgttgaccttcaccgtctggctgccgccgacgcgctccagctcgccttcctgcagcacgcgcagcagcttggcctggaacatcggcgagatctcgccgatctcgtcgaggaacagggtgccgccgtcggcctgttcgaaacgtcccttgcgctgcttcacggcgccggtgaaggcgcctttctcgtgaccgaacagttccgattcgagcagggtttccggtagcgcggcgcagttcaggcgtaccagcggctggtgagcgcgcggtgagttgtagtggatggcgctggcgatcagctccttgccggtgccggattcgccgaggatcagcac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caacgtacgcaacgacggcacctaccccggcaaggacaccggcgccctgctgatgcgccgcggcgcggtgggttgcgtctacgacgtcggcacctacctgcaggatcagctgatctaccgcgtgcatttcctcgatcagggctgcacggtgggctgccgcgaggaggagctgattcccgcgtcggacccttggatacccaacctgttcgagttccgcgaccaggtggtcgccacccgcagcctggccgtgcgcggcgaggtggtggtggagcagggccgcaccggcagcatcgagaaggtgctgcgcgacctgcccggcggcatccagtaccacgtctatttcggcgacggccgggtgcttcaggtgcccgagacgagcctggcctgggccgacgcgcaggcgggagacgagcatgagcattgatctggtcatcggcaaggatgcccgctaccagctgctgaaggtcgcccacgagcgtttcggctgtgccccggccgccctcagttcgcaacagcgtgaacaggccgagcgcatcatcggtcgccagctgcagctggagaacgccgtgctgcacagcgccgaggcctgcggtgtggtgatcccggacgagcaggtcgccgatgcctgggccgagatcgccgcccgctacgaggacccgctcgcgctgcacaaggcgctagacgacagtggtctggacgaagccggcctgcgccagctgctggcccgcgaactcaaggtcgaaacggtgctgcagcgtgtctgcgccgggctgccggaaatcaccgatacagatgtcagcctgtactacttcaatcatccggagcgcttcgtgcggcccgccacgcgactggcgcgacagatcctgattaccgtcaacgaggatttcccggaaaacagccggaccagcgcttggcgccgcatcaacctgatcgccgagcgcctgctgcgcaagccgcagcgcttcgccgagcaggcgctcaagcattccgagtgcccttcggcgatggagggcggaagcctcggcctgatacgccccggcgtgctctatccgcagctggaagcctgcctgttcgccttgcgcgcaggcgagatcggcccggtggtggagacgccactgggctttcacctgctgttctgcgaggagatccatccggcgggccatttgtcgctgcaggaggtcttgccgcacctgcgcgagaagctccgcgcccgtcaatacgagcggcaccagcgcgcatggctggccggtttgctgcagtccgccccaacctcaccggagtcgctgccatgactgataccgacaagccctgctgttcgttctgcggcgcggaaaaatcaccgacggtacccttgatcgcgggtaacgaaggccggatctgcgaggcctgcgtcaagctggcccaccaggtggtgaccagctgggggcagcggcgccaggcccagcaactggcgccgcaactgctcacgccggcggcctacatgcagcatctggacgagtcggtgatcggccaggacgaggccaaggaaaccctggcggtggcggtctacaaccactacctgcgcctgctcaactgcacccgcgagccggtctgccaactgggcggaacggtcgagctggagaagtccaacatcctcatggccggcccttcgggcaccggcaagaccctgctggtgcgcaccctggcgcgcatcctcggcgtgcccttcgcctcggctgatgccaccaccctgacccaggccggctacgtcggcgacgacgtcgacagcatcatcgcccgcctgctggaagccgccggtggcgatgtgcagaaggcgcaatggggcatcgtctatatcgacgaggtggacaagctggcacggcgtggcgggggcggcacggcggtgcgcgacatctccggcgaaggcgtgcagcaggcgctgctcaagctggtcgagggtagcgaggtgcgcatcggcaaggggggccggcgtggcgaacacggcgaggagcaggtggtggatacgcgcaacatcctgttcatcgccggtggcgcctttccgggcctggaaaccctggtcggcagccgtgtgcatccgcgtggcagcgcgatcggcttccatgcgcggccgcagcagcaggcaccgtcgatcaacgagctgctggcggcgctgctgccggacgacctccatgagttcgggctgatccccgagttcatcggtcgcttcccgatcatcaccttcctccgcgagttggaccacgcgacgctgctgcgcatcctcagcgaaccgcgcaatgcgctggtcaagcagtaccagcaactgttcgcctaccagggcgtgaagctggagttcagcgaggcggcgctcggccacatagccgaccaggcgctgctgcgccgcaccggcgcgcgcgggctgcgcgcggtcatggagagcgcgctgcagcgcaccatgttcgagatgccggcgcagccgcagctgcgcagttgcctgctcgacctcgacgaggagggccgcgaactggtggtgctcaggcagttcgacgagtatgccgaagcgcaacctgccgacagccgggcggccgcggcgtcctggcagcgttccctgctggtggtggatggctagtgtcgcattgccgacagcggcatgccgctgtcggcggccggtttgtgtggtttgcgacaggtaatgttcatgaaaaggctttgttttcattggcttataagaatccagcggctggcgtgtttcctgctatgagtcttttgccgagtgggtatgtgggcccgcggtgtttcattcatccaaacagcaatgaggtggcgtgatggccaggatcggacttttcttcggcagcaacacgggcaagacgcgcaaggtcgccaagatgatcaagaagcgcttcgacgacgacaccctggctgatccgctcaacgtcaaccgcacgagcgccgcagacttcgccggctattcgcacctgatcctcggcacgccgaccctgggcgagggcttgctgccggggctgagcgccgattgcgagaacgaaagctgggaggaattcctgccgcagatcgaggggctggatttcaccggcaagaccgtggccatcttcggcctcggcgatcaggtcggctacgccgacgagtttctcgatgcgatgggcgaactgcacgaattcttcagcgagcgcggcgccaccatggtcggcgagtggccgaccacgggctacgaattcacccactccgaagcggtggtggacggcaagttcgtcgggctggcgctggacttggacaaccagagcaacctcaccgaggagcggctgggcgcctggttgcgacagatcgctccggccttcgaactgccgctgtgaccatgcgttgagcttcgctgcacgcccggccccgacctacgcctcgtaatccgtaggttgggttgatacgcgcagcatcgaagcccaacgcgctccgaagcgcagctcggcggcgatctgtgcgaaccgttgcccggcggccgtgGGCGGAGTAGCGTGATCGCGAACCGAGGAAGGAGATTCGCC (SEQ ID NO. 6)GGCGTATCACGAGGCCCTTTCGTCTTCACCTCGAGAAAATTTATCAAAAAGAGTGTTGACTTGTGAGCGGATAACAATGATACTTAGATTCAATTGTGAGCGGATAACAATTTCACACATCTAGAGCTAATCTTCTCGTACTCATGACGCAAGTAATGAACACGATTAACATCGCTAAGAACGACTTCTCTGACATCGAACTGGCTGCTATCCCGTTCAACACTCTGGCTGACCATTACGGTGAGCGTTTAGCTCGCGAACAGTTGGCCCTTGAGCATGAGTCTTACGAGATGGGTGAAGCACGCTTCCGCAAGATGTTTGAGCGTCAACTTAAAGCTGGTGAGGTTGCGGATAACGCTGCCGCCAAGCCTCTCATCACTACCCTACTCCCTAAGATGATTGCACGCATCAACGACTGGTTTGAGGAAGTGAAAGCTAAGCGCGGCAAGCGCCCGACAGCCTTCCAGTTCCTGCAAGAAATCAAGCCGGAAGCCGTAGCGTACATCACCATTAAGACCACTCTGGCTTGCCTAACCAGTGCTGACAATACAACCGTTCAGGCTGTAGCAAGCGCAATCGGTCGGGCCATTGAGGACGAGGCTCGCTTCGGTCGTATCCGTGACCTTGAAGCTAAGCACTTCAAGAAAAACGTTGAGGAACAACTCAACAAGCGCGTAGGGCACGTCTACAAGAAAGCATTTATGCAAGTTGTCGAGGCTGACATGCTCTCTAAGGGTCTACTCGGTGGCGAGGCGTGGTCTTCGTGGCATAAGGAAGACTCTATTCATGTAGGAGTACGCTGCATCGAGATGCTCATTGAGTCAACCGGAATGGTTAGCTTACACCGCCAAAATGCTGGCGTAGTAGGTCAAGACTCTGAGACTATCGAACTCGCACCTGAATACGCTGAGGCTATCGCAACCCGTGCAGGTGCGCTGGCTGGCATCTCTCCGATGTTCCAACCTTGCGTAGTTCCTCCTAAGCCGTGGACTGGCATTACTGGTGGTGGCTATTGGGCTAACGGTCGTCGTCCTCTGGCGCTGGTGCGTACTCACAGTAAGAAAGCACTGATGCGCTACGAAGACGTTTACATGCCTGAGGTGTACAAAGCGATTAACATTGCGCAAAACACCGCATGGAAAATCAACAAGAAAGTCCTAGCGGTCGCCAACGTAATCACCAAGTGGAAGCATTGTCCGGTCGAGGACATCCCTGCGATTGAGCGTGAAGAACTCCCGATGAAACCGGAAGACATCGACATGAATCCTGAGGCTCTCACCGCGTGGAAACGTGCTGCCGCTGCTGTGTACCGCAAGGACAAGGCTCGCAAGTCTCGCCGTATCAGCCTTGAGTTCATGCTTGAGCAAGCCAATAAGTTTGCTAACCATAAGGCCATCTGGTTCCCTTACAACATGGACTGGCGCGGTCGTGTTTACGCTGTGTCAATGTTCAACCCGCAAGGTAACGATATGACCAAAGGACTGCTTACGCTGGCGAAAGGTAAACCAATCGGTAAGGAAGGTTACTACTGGCTGAAAATCCACGGTGCAAACTGTGCGGGTGTCGACAAGGTTCCGTTCCCTGAGCGCATCAAGTTCATTGAGGAAAACCACGAGAACATCATGGCTTGCGCTAAGTCTCCACTGGAGAACACTTGGTGGGCTGAGCAAGATTCTCCGTTCTGCTTCCTTGCGTTCTGCTTTGAGTACGCTGGGGTACAGCACCACGGCCTGAGCTATAACTGCTCCCTTCCGCTGGCGTTTGACGGGTCTTGCTCTGGCATCCAGCACTTCTCCGCGATGCTCCGAGATGAGGTAGGTGGTCGCGCGGTTAACTTGCTTCCTAGTGAAACCGTTCAGGACATCTACGGGATTGTTGCTAAGAAAGTCAACGAGATTCTACAAGCAGACGCAATCAATGGGACCGATAACGAAGTAGTTACCGTGACCGATGAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGAGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGAATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAGAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCCTATTCAGACGCGCTTGAACCTGATGTTCCTCGGTCAGTTCCGCTTACAGCCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTCGGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTAAcagatctcatcaccatcaccatcactaagcttaattagctgagcttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatccaagctagcttggcgagatccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcaggccaaccagataagtgaaatctagttccaaactattttgtcatttttaattttcgtattagcttacgacgctacacccagttcccatctattttgtcactcttccctaaataatccttaaaaactccatttccacccctcccagttcccaactattttgtccgcccacagcggggcatttttcttcctgttatgtttgggcgctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaacaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagccggttgtcagccgttaagtgttcctgtgtcactcaaaattgctttgagaggctctaagggcttctcagtgcgttacatccctggcttgttgtccacaaccgttaaaccttaaaagctttaaaagccttatatattcttttttttcttataaaacttaaaaccttagaggctatttaagttgctgatttatattaattttattgttcaaacatgagagcttagtacgtgaaacatgagagcttagtacgttagccatgagagcttagtacgttagccatgagggtttagttcgttaaacatgagagcttagtacgttaaacttgagagcttagtacgtgaaacatgagagcttagtacgtactatcaacaggttgaactgcccatgttctttcctgcgttatcagagcttatcggccagcctcgcagagcaggattcccgttgagcaccgccaggtgcgaataagggacagtgaagaaggaacacccgctcgcgggtgggcctacttcacctatcctgcccggctgacgccgttggatacaccaaggaaagtctacacgaaccctttggcaaaatcctgtatatcgtgcgaaaaaggatggatataccgaaaaaatcgctataatgaccccgaagcagggttatgcagcggaaagtataccttaacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctgcggttgagtaataaatggatgccctgcgtaagcgggtgtgggcggacaataaagtcttaaactgaacaaaatagatctaaactatgacaataaagtcttaaactagacagaatagttgtaaactgaaatcagtccagttatgctgtgaaaaagcatactggacttttgttatggctaaagcaaactcttcattttctgaagtgcaaattgcccgtcgtattaaagaggggcgtggggttcgaggtcgacggtatcgataagctagcttaattagctgagcttggaagtacctattccgaagttcctattctctagaaagtataggaacttcagcggaaaaggacaattgtcTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAGTGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCCACAATGGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGACGGCGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCGTGCAGGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGAATCCACAGGACGGGTGTGGTCGCCATGATCGCGTAGTCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGACTGGGCGGCGGCCAAAGCGGTCGGACAGTGCTCCGAGAACGGGTGCGCATAGAAATTGCATCAACGCATATAGCGCTAGCAGCACGCCATAGTGACTGGCGATGCTGTCGGAATGGACGATATCCCGCAAGAGGCCCGGCAGTACCGGCATaaccaagcctatgcctacagcatccagggtgacggtgccgaggatgacgatgagcgcattgttagatttcatacacggtgcctgactgcgttagcaatttaactgtgataaactaccgcattacagtttatcgatgataagctgtcaagaagttcctattccgaagttcctattctctagaaagtataggaacttctgcatttacgttgacaccatAATAAAAAAGCCCCCGGAATGATCTTCCGGGGGCtcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagactggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctatcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcctcatgggagtaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccaccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtcatataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcaccattcgatggtgtcaacgtaaatgcatgccgcttcgccttcgcgcgcgaattgcaggtaccatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaata

The modified bacteria described herein are capable of colonizing a hostplant. In certain cases, the modified bacteria can be applied to theplant, by foliar application, foliar sprays, stem injections, soildrenches, immersion, root dipping, seed coating or encapsulation usingknown techniques.

Successful colonization can be confirmed by detecting the presence ofthe bacterial population within the plant. For example, after applyingthe bacteria to the seeds, high titers of the bacteria can be detectedin the roots and shoots of the plants that germinate from the seeds. Inaddition, significant quantities of the bacteria can be detected in therhizosphere of the plants. Therefore, in one embodiment, the endophyticmicrobe population is disposed in an amount effective to colonize theplant. Colonization of the plant can be detected, for example, bydetecting the presence of the endophytic microbe inside the plant. Thiscan be accomplished by measuring the viability of the microbe aftersurface sterilization of the seed or the plant: endophytic colonizationresults in an internal localization of the microbe, rendering itresistant to conditions of surface sterilization.

In some cases, the modified bacteria is mixed with an agriculturallysuitable or compatible carrier. The carrier can be a solid carrier orliquid carrier. The carrier may be any one or more of a number ofcarriers that confer a variety of properties, such as increasedstability, wettability, or dispersability. Wetting agents such asnatural or synthetic surfactants, which can be nonionic or ionicsurfactants, or a combination thereof can be included in a compositionof the invention. Water-in-oil emulsions can also be used to formulate acomposition that includes the modified bacteria of the presentinvention. Suitable formulations that may be prepared include wettablepowders, granules, gels, agar strips or pellets, thickeners, and thelike, microencapsulated particles, and the like, liquids such as aqueousflowables, aqueous suspensions, water-in-oil emulsions, etc. Theformulation may include grain or legume products, for example, groundgrain or beans, broth or flour derived from grain or beans, starch,sugar, or oil.

In some embodiments, the agricultural carrier may be soil or plantgrowth medium. Other agricultural carriers that may be used includefertilizers, plant-based oils, humectants, or combinations thereof.Alternatively, the agricultural carrier may be a solid, such asdiatomaceous earth, loam, silica, alginate, clay, bentonite,vermiculite, seed cases, other plant and animal products, orcombinations, including granules, pellets, or suspensions. Mixtures ofany of the aforementioned ingredients are also contemplated as carriers,such as but not limited to, pesta (flour and kaolin clay), agar orflour-based pellets in loam, sand, or clay, etc. Formulations mayinclude food sources for the cultured organisms, such as barley, rice,or other biological materials such as seed, plant parts, sugar canebagasse, hulls or stalks from grain processing, ground plant material orwood from building site refuse, sawdust or small fibers from recyclingof paper, fabric, or wood. Other suitable formulations will be known tothose skilled in the art.

In one embodiment, the formulation can comprise a tackifier or adherent.Such agents are useful for combining the modified bacteria with carriersthat can contain other compounds (e.g., control agents that are notbiologic), to yield a coating composition. Such compositions help createcoatings around the plant or seed to maintain contact between themicrobe and other agents with the plant or plant part. In oneembodiment, adherents are selected from the group consisting of:alginate, gums, starches, lecithins, formononetin, polyvinyl alcohol,alkali formononetinate, hesperetin, polyvinyl acetate, cephalins, GumArabic, Xanthan Gum, Mineral Oil, Polyethylene Glycol (PEG), Polyvinylpyrrolidone (PVP), Arabino-galactan, Methyl Cellulose, PEG 400,Chitosan, Polyacrylamide, Polyacrylate, Polyacrylonitrile, Glycerol,Triethylene glycol, Vinyl Acetate, Gellan Gum, Polystyrene, Polyvinyl,Carboxymethyl cellulose, Gum Ghatti, and polyoxyethylene-polyoxybutyleneblock copolymers.

The formulation can also contain a surfactant. Non-limiting examples ofsurfactants include nitrogen-surfactant blends such as Prefer 28(Cenex), Surf-N(US), Inhance (Brandt), P-28 (Wilfarm) and Patrol(Helena); esterified seed oils include Sun-It II (AmCy), MSO (UAP),Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); andorgano-silicone surfactants include Silwet L77 (UAP), Silikin (Terra),Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis) andCentury (Precision).

In certain cases, the formulation includes a microbial stabilizer. Suchan agent can include a desiccant. As used herein, a “desiccant” caninclude any compound or mixture of compounds that can be classified as adesiccant regardless of whether the compound or compounds are used insuch concentrations that they in fact have a desiccating effect on theliquid inoculant. Such desiccants are ideally compatible with themodified bacteria used, and should promote the ability of the microbialpopulation to survive application on the seeds and to survivedesiccation. Examples of suitable desiccants include one or more oftrehalose, sucrose, glycerol, and methylene glycol. Other suitabledesiccants include, but are not limited to, non reducing sugars andsugar alcohols (e.g., mannitol or sorbitol).

The formulations may also include one or more agents such as afungicide, an antibacterial agent, an herbicide, a nematicide, aninsecticide, a plant growth regulator, a rodenticide, and a nutrient.Such agents are ideally compatible with the agricultural seed orseedling onto which the formulation is applied (e.g., it should not bedeleterious to the growth or health of the plant).

When the formulation is a liquid solution or suspension, the modifiedbacteria can be mixed or suspended in aqueous solutions. Suitable liquiddiluents or carriers include aqueous solutions, petroleum distillates,or other liquid carriers.

A formulation that is a solid composition can be prepared by dispersingthe modified bacteria in or on an appropriately divided solid carrier,such as peat, wheat, bran, vermiculite, clay, talc, bentonite,diatomaceous earth, fuller's earth, or pasteurized soil. When suchformulations are used as wettable powders, biologically compatibledispersing agents such as nonionic, anionic, amphoteric, or cationicdispersing and emulsifying agents can be used.

Solid carriers useful in aspects of the invention include, for example,mineral carriers such as kaolin clay, pyrophyllite, bentonite,montmorillonite, diatomaceous earth, acid white soil, vermiculite, andpearlite, and inorganic salts such as ammonium sulfate, ammoniumphosphate, ammonium nitrate, urea, ammonium chloride, and calciumcarbonate. Also, organic fine powders such as wheat flour, wheat bran,and rice bran may be used. The liquid carriers include vegetable oilssuch as soybean oil and cottonseed oil, glycerol, ethylene glycol,polyethylene glycol, propylene glycol, polypropylene glycol, etc.

The modified bacteria herein can be combined with one or more of theagents described herein to yield a formulation suitable for combiningwith a plant, a seed or seedling. The modified bacteria can be obtainedfrom growth in culture, for example, using a synthetic growth medium. Inaddition, the microbe can be cultured on solid media, for example onpetri dishes, scraped off and suspended into the preparation. Microbesat different growth phases can be used. For example, microbes at lagphase, early-log phase, mid-log phase, late-log phase, stationary phase,early death phase, or death phase can be used.

In some embodiments the invention also includes containers or equipmentwith the modified bacteria, with or without the plants, seeds orseedlings. For instance, the invention may include a bag comprising atleast 1,000 seeds having modified bacteria. The bag further comprises alabel describing the seeds and/or said modified bacteria.

The population of seeds may be packaged in a bag or container suitablefor commercial sale. Such a bag contains a unit weight or count of theseeds comprising the modified bacteria as described herein, and furthercomprises a label. In one embodiment, the bag or container contains atleast 1,000 seeds, for example, at least 5,000 seeds, at least 10,000seeds, at least 20,000 seeds, at least 30,000 seeds, at least 50,000seeds, at least 70,000 seeds, at least 80,000 seeds, at least 90,000seeds or more. In another embodiment, the bag or container can comprisea discrete weight of seeds, for example, at least 1 lb, at least 2 lbs,at least 5 lbs, at least 10 lbs, at least 30 lbs, at least 50 lbs, atleast 70 lbs or more. The bag or container comprises a label describingthe seeds and/or said modified bacteria. The label can containadditional information, for example, the information selected from thegroup consisting of: net weight, lot number, geographic origin of theseeds, test date, germination rate, inert matter content, and the amountof noxious weeds, if any. Suitable containers or packages include thosetraditionally used in plant seed commercialization.

A substantially uniform population of seeds comprising the modifiedbacteria is provided in other aspects of the invention. In someembodiments, at least 10%, for example, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 90%, at least 95% or more of the seeds in thepopulation, contains the modified bacteria in an amount effective tocolonize a plant. In other cases, at least 10%, for example, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 90%, at least 95% or more ofthe seeds in the population, contains at least 100 CFU on its surface,for example, at least 200 CFU, at least 300 CFU, at least 1,000 CFU, atleast 3,000 CFU, at least 10,000 CFU, at least 30,000 CFU, at least100,000 CFU, at least 300,000 CFU, or at least 1,000,000 CFU per seed ormore.

Alternatively a substantially uniform population of plants is provided.The population comprises at least 100 plants, for example, at least 300plants, at least 1,000 plants, at least 3,000 plants, at least 10,000plants, at least 30,000 plants, at least 100,000 plants or more. Theplants are grown from the seeds comprising the modified bacteria asdescribed herein. The increased uniformity of the plants can be measuredin a number of different ways.

In some embodiments, there is an increased uniformity with respect tothe modified bacteria within the plant population. For example, in oneembodiment, a substantial portion of the population of plants, forexample at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 75%, at least 80%, at least90%, at least 95% or more of the seeds or plants in a population,contains a threshold number of the modified bacteria. The thresholdnumber can be at least 100 CFU, for example at least 300 CFU, at least1,000 CFU, at least 3,000 CFU, at least 10,000 CFU, at least 30,000 CFU,at least 100,000 CFU or more, in the plant or a part of the plant.Alternatively, in a substantial portion of the population of plants, forexample, in at least 1%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 90%, at least 95% or more of the plants in thepopulation, the modified bacteria that is provided to the seed orseedling represents at least 10%, least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or 100% of the total microbe population in theplant/seed.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

EXAMPLES Example 1: Nitrogen Fixation in Salmonella Using Refactored NifClusters Methodology Nitrogenase Activity Assay in Bacteria.

Acetylene reduction assay was used to measure nitrogenase activity ofbacteria in free-living conditions. Cultures were initiated byinoculating a single colony into 1 mL of LB medium with appropriateantibiotics in a 15 mL culture tube. Cultures grown with shaking at 250rpm at 37° C. for 12 h were diluted 100-fold in 1 mL of minimal mediumplus 17.1 mM NH₄Ac with appropriate antibiotics in 96-well deep wellplates. The plates were incubated with shaking at 900 rpm at 30° C. for20 h. Cultures were diluted an OD₆₀₀ of 0.5 in 2 mL of nitrogen-freeminimal medium supplemented with appropriate antibiotics and inducers in10 mL glass vials with PTFE-silicone septa screw caps (SupelcoAnalytical, Bellefonte, Pa., cat. #SU860103). Headspace in the bottleswas replaced with 100% argon gas using a vacuum manifold equipped with acopper catalyst oxygen trap. Acetylene freshly generated from CaC₂ in aBurris bottle was injected to 10% (vol/vol) into each culture vial tobegin the reaction. Cultures were allowed to grow for 20 h at 30° C.with shaking at 250 rpm, followed by quenching via the addition of 0.3mL of 4 M NaOH to each vial. Ethylene production was analyzed by gaschromatography on an Agilent 7890A GC system (Agilent Technologies, Inc.Santa Clara, Calif. USA) equipped with a PAL headspace autosampler andflame ionization detector as follows. 0.25 mL headspace preincubated to35° C. for 30 s was injected and separated for 5 min on a GS-CarbonPLOTcolumn (0.32 mm×30 m, 3 micron; Agilent) at 60° C. and a He flow rate of1.8 ml/min. Detection occurred in a FID heated to 300° C. with a gasflow of 35 ml/min H2 and 400 ml/min air. Acetylene and ethylene weredetected at 3.0 min and 3.7 min after injection, respectively. Ethyleneproduction was quantified by integrating the 3.7 min peak using AgilentGC/MSD ChemStation Software.

Seed Sterilization, Germination and Inoculation of Bacteria.

For surface-sterilization, Zea mays B73 seeds (U.S. National PlantGermplasm System, IA) first were washed with 70% ethanol and immersed in2% sodium hypochlorite solution (25% commercial bleach) for 15 min withshaking at 50 rpm and subsequently washed three times with sterilewater. Surface-sterilized seeds were placed on 1% Bacto agar platesupplemented with 1 μM of gibberellic acid (Sigma-Aldrich, MO) andincubated under dark at room temperature up to 6 days beforegermination. A regular weight germination paper (Ancor Paper Co., Mn)soaked in 10 mL of sterile water was placed on the bottom ofnitrogen-free Fahräeus agar plate. The germinated seeds weretransplanted at the top of the germination paper in Fahräeus agar plate(4 seedling/plate). After establishing rooting system for 2 days, maizeroots were flooded with 50 mL of bacteria (OD₆₀₀=1) resuspended insterile water and incubated at room temperature. Bacteria were removedby pipetting after 1 h of incubation. The plant growth was continuedunder 24 h constant light at 26° C. for additional two weeks before theassays.

Internal Colonization Assay

Two weeks post-inoculation, only plant roots were retained by removingleave and seeds from the seedling using a razor blade. To determineinternal colonization, each root was immersed in 20 mL of 1.6% sodiumhypochlorite solution (20% commercial bleach) in 50 mL falcon tube andvortexed vigorously for 1 min followed by four times washes with 25 mLof sterile water. The surface sterilized roots were vortexed in 5 mL ofPBS for 1 min following the last wash and subsequently plated on LB agarplate to quantify residual bacteria. The sterilized roots were crushedusing a mortar and pestle in 5 mL of PBS for 5 min and the extracts wereserially diluted in PBS and plated on LB agar plates with or without aselective marker to determine the presence of bacteria and the plasmidstability. The plates were incubated at 37° C. for 24 h before analyzingcolony forming unit (CFU).

Nitrogenase Activity Assay in Plants

Acetylene reduction assay was used to measure nitrogenase activity ofmaize seedlings. Two weeks post-inoculation of bacteria, the intactseedlings were transferred into 30 mL volume anaerobic culture tubes(Chemglass Life Sciences, NJ) containing 2 mL of nitrogen-free Fahräeusmedium sealed with a rubber stopper without headspace replacement. Forthe maize seedlings inoculated with the bacteria strain carrying therefactored cluster, 25 mL of 0.5 M IPTG was applied on seedling rootsgrown 13 days after inoculation of bacteria, after which the seedlingswere incubated under constant light for 12 h before transfer intoanaerobic culture tubes containing 2 mL of nitrogen-free Fahräeus mediumwith 10 mM IPTG. Acetylene freshly generated from CaC₂ in a Burrisbottle was injected to 7% (vol/vol) into each culture tube to start thereaction. The reaction was continued under a light regimen of 18 h oflight and 6 h of dark at 28° C. up to 4 days. Ethylene production wasquantified by gas chromatography. 0.5 mL of headspace was sampled andanalyzed in a manner identical to that described above.

Results

Transfer of Nif Clusters into Salmonella Strains.

Transfer of native and refactored nif clusters of Klebsiella was provento be functional in K. oxytoca M5a1 and E. coli such as K12 MG1655.However, it hasn't been shown that heterologous expression of nifclusters would be active in other enteric bacteria that can colonizeinto crop cereals. We have collected pathogenic Salmonella strains thatcan infect various hosts ranging from humans to plants. We transferrednative and refactored nif clusters into diverse Salmonella strains totest nitrogen fixation in a free living condition. Also, together withthe refactored cluster, the controller plasmid encoding a sensor andcircuit that drives the expression of the entire nif cluster in responseto IPTG was introduced into Salmonella strains.

Particularly, S. typhi strains containing the native or refactored nifcluster showed higher nitrogenase activity among diverse Salmonellastrains. Salmonella dublin, newport and pomona only exhibitednitrogenase activity from the native nif cluster to a lesser extent thanthose of the nitrogen fixing S. typhi strains (FIG. 1 ).

Internal Colonization of Zea mays B73 Roots by S. typhi

To determine whether a Salmonella strain can be a bacterial endophyte inmaize plants, we inoculated bacteria onto the roots of Zea mays B73 thatis an important commercial crop variety. S. typhi ATCC 14028 showing oneof the highest nitrogenase activity by heterologous nif expression wasselected for internal colonization assay. 14 days post-inoculation,internal colonization by S. typhi ATCC 14028 was analyzed using theroots of plant seedlings. No CFU of S. typhi ATCC 14028 was detectedafter surface sterilization of the roots. To assess internally colonizedbacteria cells, the surface sterilized roots of each plant seedling werecrushed in PBS and plated on LB plates. We detected endophyticcolonization of ˜10⁶ CFU/plant by S. typhi ATCC 14028 from the crushedroot extracts, but no CFU by E. coli MG1655 in the same setting (FIG. 2). This shows that S. typhi ATCC 14028 can colonize Zea mays B73internally.

Nitrogenase Activity in Maize Plants

14 days post-inoculation, we analyzed nitrogenase activity from theplant seedlings infected with the genetically modified S. typhi ATCC14028 strains by acetylene reduction assay. More than 30 plants fromeach group were analyzed. 18% and 51% of the plants inoculated with S.typhi ATCC 14028 carrying the native nif cluster and the refactored nifcluster, respectively, displayed increased ethylene production comparedto those plants inoculated with S. typhi ATCC 14028 expressing no nifcluster (FIG. 3 ). The refactored nif cluster as compared to the nativenif cluster resulted in less variation in acetylene reduction in plants.This suggests that the expression of refactored nif cluster is moreconsistent in our setting conferred by the synthetic controller systemthat regulates the expression of the refactored nif cluster by anexternally added inducer than that of the native nif cluster whoseregulation is still under the control of complex native biologicalsignals.

Improvement of Stability of Genetic Systems

Plasmid-based engineering of the clusters and controllers relies onplasmid stability during cell division. Such selective pressure forplasmid stability as antibiotic use can be easily applied and maintainedin an in vitro setup. However, plasmids are cured from the host bacteriaover time without selective antibiotic pressure in an in vivo setup.

In order to increase stability of the genetic system in bacteria, twoengineering strategies were used. First, we introduced a controller thatencodes an IPTG inducible T7 RNA polymerase and a selective marker intoa target genome using the mini-Tn7 system [Choi, K. H., (2005). ATn7-based broad-range bacterial cloning and expression system. Naturemethods, 2(6), 443-448.]. It has been demonstrated that thetransposition with the mini-Tn7 system is broad-host range andsite-specific. Genome integration occurs at the Tn7 attachment site(attTn7) located downstream of the essential gene glmS. Salmonellacontains a single glmS gene that ensures a single-copy insertion of anintroduced genetic system. A new controller plasmid pR6K-T7RW designedfor genome integration consists of a T7 RNA polymerase and a selectionmarker flanked by two Tn7 ends (Tn7L and Tn7R). To minimize interferenceby transcriptional read-through from the upstream glmS expression, aconstitutive promoter-driven selection marker and a sensor protein ladare oriented opposite to the glmS. A T7 RNA polymerase read-through wasblocked by a terminator between the device and the genome. Wetransformed a controller plasmid pR6K-T7RW and a helper plasmid pTNS3encoding the TnsABCD transposase into Salmonella ATCC14028. Theinsertion site of a controller device was verified by PCR. We identifiedthat the device is integrated 25 bp downstream of the glmS stop codon inSalmonella. We tested plasmid stability based on a selective marker inthe internally colonized Salmonella strains containing either agenome-based controller or a plasmid-based controller two weeks afterinoculation of germinated maize seeds. There was no marker loss from thegenome-based system, whereas only about 20% of strains from theplasmid-based system were retained on the plates supplemented withantibiotics, indicating that the controller device on the Salmonellagenome was stable without selective pressure over two weeks in the plantseedlings (FIG. 4A).

The nif clusters were constructed on a broad-host range plasmid pBBR1such that the optimal expression levels of the nif genes in diversecontexts can be rapidly accessed by swapping genetic parts of theclusters on a plasmid. To keep the versatility and engineerablity of aplasmid-based nif system, we sought to explore an alternative togenome-based engineering while ensuring the stability of the nifclusters on the plasmid. The partitioning system encoded by the two paroperons (parCBA and parDE) contributes to stable maintenance of aplasmid RK2 [Easter, C. L., Schwab, H., & Helinski, D. R. (1998). Roleof the parCBA operon of the broad-host-range plasmid RK2 in stableplasmid maintenance. Journal of bacteriology, 180(22), 6023-6030.].However, the transferability of the function of the RK2 par system hasnot been tested on other types of plasmids. We integrated the RK2 parsystem into the nif plasmids built upon a plasmid pBBR1 and analyzedplasmid stability in the Salmonella strain from the colonized roots. Thenif plasmid stability without the par system decreased to 4% in theabsence of a selective pressure after 14 days of inoculation into theplants. On the other hand, adding the par system on the nif plasmidsresulted in plasmid stability of 96% under the identical conditions,which suggesting the RK2 par system works as a module to improve thestability of other plasmid types (FIG. 4 B). These engineering effortscan be modular standards as a means to provide the stability of complexmultigene systems in the bacteria that are supposed to be released intothe environment.

REFERENCES

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety.

1-12. (canceled)
 13. A method, comprising delivering a modifiednon-pathogenic bacteria having exogenous genes for enabling plantendosymbiosis to a cereal plant, or to soil where a cereal plant or seedis growing or is to be planted.
 14. The method of claim 13, wherein thenon-pathogenic bacteria is E. coli.
 15. The method of claim 14, whereinthe genes encode effectors or apparatus for a secretion system.
 16. Themethod of claim 15, wherein the apparatus for a secretion system is type3 secretion system (T3SS).
 17. The method of claim 13, wherein theexogenous gene includes a controller.
 18. The method of claim 17,wherein the controller is a nucleic acid encoding an IPTG inducible T7RNA polymerase.
 19. The method of claim 17, wherein the controller is apartitioning system encoded by the two par operons (parCBA and parDE).20. The method of claim 17, wherein the partitioning system is a RK2 parsystem.
 21. A composition, comprising: (a) an agriculturally suitablecarrier; and (b) a gamma-proteobacteria having an exogenous nif clusterpresent on or in the agriculturally suitable carrier.
 22. Thecomposition of claim 21, wherein the gamma-proteobacteria is aSalmonella typhimurium or E. coli.
 23. The composition of claim 21,wherein the nif cluster is a native nif cluster.
 24. The composition ofclaim 21, wherein the nif cluster is a refactored nif cluster.
 25. Thecomposition of claim 21, further comprising an exogenous gene encoding aplant growth-stimulating peptide.
 26. The composition of claim 21,wherein the agriculturally suitable carrier is selected from the groupconsisting of seeds, seed coats, granular carriers, soil, solidcarriers, liquid slurry carriers, and liquid suspension carriers. 27.The composition of claim 21, wherein the agriculturally suitable carrierincludes a wetting agents, a synthetic surfactant, a water-in-oilemulsion, a wettable powder, granules, gels, agar strips or pellets,thickeners, microencapsulated particles, or liquids such as aqueousflowables or aqueous suspensions.
 28. The composition of claim 21,wherein the exogenous nif cluster includes a controller.
 29. Thecomposition of claim 28, wherein the controller is a nucleic acidencoding an IPTG inducible T7 RNA polymerase.
 30. The composition ofclaim 28, wherein the controller is a partitioning system encoded by thetwo par operons (parCBA and parDE).
 31. The composition of claim 28,wherein the partitioning system is a RK2 par system.
 32. A seed orseedling of a cereal plant having a modified bacteria associated with anexternal surface of the seed or seedling; wherein said modified bacteriacomprises a refactored exogenous nif cluster.
 33. (canceled)
 34. Theseed or seedling of claim 32, wherein the nif cluster is a native nifcluster.
 35. The seed or seedling of claim 32, wherein the nif clusteris a refactored nif cluster.
 36. The seed or seedling of claim 32,wherein the modified bacteria is a gamma-proteobacteria.
 37. The seed orseedling of claim 36, wherein the gamma-proteobacteria is a Salmonellatyphimurium.
 38. The seed or seedling of claim 37, wherein theSalmonella typhimurium strain is selected from SL1344, LT2, and DW01.39. The seed or seedling of claim 32, wherein the modified bacteria is aE. coli, optionally of strain H7:0157.
 40. The seed or seedling of claim32, wherein the nif cluster is a Klebsiella wild-type nif cluster, aPseudomonas Stutzi nif cluster, or a Paenibacillus cluster.
 41. The seedor seedling of claim 32, wherein the cereal plant is selected fromwheat, rye, barley, triticale, oats, millet, sorghum, teff, fonio,buckwheat, quinoa, corn and rice.
 42. The seed or seedling of claim 32,further comprising an exogenous gene encoding a plant growth-stimulatingpeptide.
 43. The seed or seedling of claim 42, wherein the exogenousgene encoding the plant growth-stimulating peptide is regulated by atype 3 secretion system (T3SS).
 44. A cereal plant having a modifiedbacteria in the plant, wherein the modified bacteria has an exogenousnif cluster.
 45. The cereal plant of claim 44, wherein the nif clusteris a refactored nif cluster.
 46. The cereal plant of claim 44, furthercomprising an exogenous gene encoding a plant growth-stimulatingpeptide.
 47. The cereal plant of claim 46, wherein the exogenous geneencoding the plant growth-stimulating peptide is regulated by a type 3secretion system (T3SS).
 48. The cereal plant of claim 46, wherein theexogenous gene encodes effectors or apparatus for a secretion system.49. The cereal plant of claim 47, wherein the exogenous gene is in rootor stem tissues of the plant.
 50. A method of providing a corn plantwith fixed nitrogen from atmospheric nitrogen, comprising: delivering atransgenic bacteria having a refactored exogenous nif cluster to a cornplant, or to soil where a corn plant or seed is growing or is to beplanted.
 51. The method of claim 37, wherein a non-transgenic bacteriahaving a same species as said transgenic bacteria lacks a nif cluster.